430 results for Undergraduate

  • Novel Methods for the Rapid Identification and Susceptibility Testing of Blood Culture Isolates

    Robinson, Andrew Mark (2014)

    Undergraduate thesis
    University of Otago

    The increasing emergence of antimicrobial resistance, such as that mediated by extended-spectrum β-lactamase (ESBL)-producing gram-negative bacteria, make it more likely that patients with sepsis and bloodstream infections (BSI) will receive ineffective empirical treatment. Rapid identification of disease causing agents, coupled with early detection of antimicrobial resistance facilitates the optimisation of essential treatment decisions. Matrix-assisted laser desorption-ionization time of flight (MALDI-ToF) mass spectrometry has recently been applied to the identification of microorganisms directly from blood cultures, reducing the identification process by up to 24-hours. This study sought (i) to determine the optimal method for the rapid identification of isolates directly from blood cultures and (ii) to develop a rapid method to detect β-lactamase-mediated resistance to extended spectrum cephalosporins directly from blood cultures. Two in-house methods for sample preparation were optimized and compared to a commercially available method. Using the conventional scoring criteria, the differential centrifugation protocol correctly identified 86.8% and 67.9% of clinical isolates at the genus- and species- level. This was compared to a quicker method using Sodium dodecyl sulphate (SDS) to mediate blood cell lysis, which correctly identified 83.0% and 62.3% of clinical isolates to the genus- and species- level. Both methods performed similarly to the more expensive commercial method. Results also suggested that the scoring criteria could be altered to increase the number of species-level identification while maintaining accuracy, achieving up to 90.3% species level identifications. To rapidly detect β-lactamase-mediated resistance to extended spectrum cephalosporins, a high-performance liquid chromatography (HPLC) assay was developed and optimized to detect resistance directly from growth-positive blood cultures. With a 1-hour incubation of bacteria with cefotaxime, resistance could be detected with 95.5% sensitivity and 88.9% specificity. This method was better at detecting resistance mediated by group 1 and 9 CTX-M ESBLs, with reduced sensitivity for the detection of resistance mediated by AmpC β-lactamases. Further research is required to investigate additional markers that could improve the detection of other β-lactamases. Both of these methods could be rapidly integrated into the diagnostic microbiology laboratory, thus reducing the time to effective narrow spectrum antimicrobial therapy, and potentially improving patient outcomes and reducing the spread of antimicrobial resistance.

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  • Comparing subpopulations of gonadotropin-releasing hormone (GnRH) neurons with viral mediated cell-filling

    Marshall, Christopher Joseph (2014)

    Undergraduate thesis
    University of Otago

    Gonadotropin-releasing hormone (GnRH) neurons are the central regulators of reproductive function in all mammals. The cell bodies of GnRH neurons are unique in that they are not confined to a discrete nucleus, but rather exist as a scattered continuum of cells throughout the basal forebrain. In rodent species, most of these GnRH neurons reside within three anatomical divisions of the brain: the medial septum (MS), the rostral preoptic area (rPOA) and the anterior hypothalamic area (AHA). Typically, these neurons are thought of as one large homogenous group, serving similar functions, however, recent anatomical and functional evidence suggests that this is not the case. One way to distinguish subsets of neurons is by their patterns of projection throughout the brain. Until now, the projection patterns of GnRH neurons have only ever been assessed for the population as a whole, due to the lack of ability to distinguish subdivisions from one another. The recent development of novel transgenic tools has enabled us to visualize GnRH neurons and their projections in their anatomical subdivisions of the MS, rPOA or AHA for the first time. An adenovirus containing a transgene for enhanced, farnesylated green fluorescent protein (Ad-iZ/EGFPf) was injected intracranially at stereotaxic coordinates for the MS (n=4), rPOA (n=6) or AHA (n=4) into female transgenic GnRH-Cre mice. Using this approach, Ad-iZ/EGFPf was specifically targeted to GnRH neurons in each region of interest, in order to “fill” these cells to the most distal ends of their projections. The first aim of this project was to assess how accurately GnRH neurons in MS, rPOA and AHA could be specifically targeted. Injections filled between 5 and 20 cells in most animals with accurate injection sites. In animals that received MS injections, more GnRH neurons in the MS were filled than in the rPOA (P < 0.05) and AHA (P < 0.05). Similarly, animals that received rPOA injections had more filled cells in the rPOA compared with the MS (P < 0.0001) and AHA (P < 0.001). In animals injected in the AHA there was no significant difference in the number of filled cells in the AHA compared with the MS and rPOA. In wild-type controls (n=3) and animals that received off-target injections (n=3), no filled GnRH neurons or projections were present. In the second aim of this project the distribution of projections from Ad-iZ/EGFPf filled GnRH neurons residing in the MS, rPOA and AHA were mapped. Across all animals, GnRH neuron fibre projections that were positive for GFP were found in 120 different regions of the brain, including nuclei, subnuclei and white matter tracts. These regions were found across several major brain divisions, in the hypothalamus, septum, thalamus, cerebral cortex, pallidum, striatum, amygdala, hippocampus and midbrain. The broad distribution of GnRH neuron projections highlights the diverse functions that these neurons are potentially influencing within the brain, as well as the power of the viral cell-filling approach used to visualize the full extent of these neurons. In the third aim, the projection patterns from GnRH neurons in the MS, rPOA and AHA were compared. Remarkably, 60% of the brain regions that contained fibre projections only did so from one or a combination of any two subpopulations of GnRH neurons, indicating that the projection patterns of these subdivisions is not homogenous. Notably, fibre projections in the vomeronasal amygdala originated exclusively from GnRH neurons in the AHA. Areas involved with olfactory processing likewise only received projections from MS and AHA GnRH neurons. Surprisingly, the largest division of GnRH neurons, the rPOA, had the most confined pattern of projection, but projected robustly to the median eminence suggesting a primarily hypophysiotropic role. For the first time, it has been possible to interrogate the projection patterns of anatomical subdivisions of GnRH neurons, which has revealed that they are far from homogenous. Overall, these results provide strong support for the existence of GnRH neuron subpopulations, highlighting that these neurons should be treated as similar but separate entities. Identifying GnRH neural subpopulations and delineating their respective roles could have wide applications, from increasing reproductive success in livestock, to teasing apart the ongoing mysteries surrounding infertility in humans.

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  • Understanding Batten disease: CLN5 expression in CLN6 deficient ovine neural cultures.

    McIntyre, Kristina (2014)

    Undergraduate thesis
    University of Otago

    Neuronal Ceroid Lipofuscinoses (NCL) are a group of debilitating and fatal neurodegenerative diseases of childhood resulting from progressive brain atrophy. The human and ovine variant late infantile CLN6 (ceroid lipofuscinosis protein) forms of NCL result from mutations encoding the endoplasmic reticulum transmembrane protein CLN6. Mutations encoding the soluble lysosomal protein CLN5 also cause an NCL in humans and sheep. The functions of these proteins are not understood, however previous research suggests a relationship between them (Lyly et al., 2009). In CLN6-/- ovine neural tissue, CLN5 protein expression was reduced (McIntyre, K.M., summer studentship unpublished observations, 2013/2014). The aim of this study was to investigate this potential interaction in the ovine model of CLN6 NCL. In CLN6-/- and CLN6+/- control secondary ovine cultures derived from primary ovine neural cultures, concentrations of CLN5 mRNA were assessed by relative quantitative polymerase chain reaction (qPCR). Protein expression was assessed by 3, 3 diaminobenzidine (DAB) immunocytochemistry, immunofluorescence and western blotting techniques. To detect CLN5 protein, cultures were transduced with CLN5 lentivirus (pCDH.MND.CLN5 (VSVG)). Proteasome inhibitor MG132 was applied for 2 and 4 hours to determine whether the reduction in CLN5 was due to ERAD (Endoplasmic Reticulum Associated-Protein Degradation). Absence of GFAP (Glial Fibrillary Acidic Protein) and MAP2 (Microtubule-associated protein 2) immunofluorescence in cultures indicated a cell population devoid of astrocytes and neurons respectively. Relative to ATPase and RPLPO (Large Ribosomal Protein) (M-value 0.936), no statistical difference in CLN5 mRNA concentration was found between CLN6-/- and control cultures (p = 0.68, n = 3). DAB immunocytochemistry showed low CLN5 protein expression in non-transduced cultures; reduced CLN5 protein expression was not evident. In immunofluorescence studies, no significant difference in relative fluorescent intensity was seen in transduced cultures (p = 0.75, n = 3). Western blot analysis of overexpressed CLN5 protein between CLN6-/- and control cultures was inconclusive. In non-transduced cultures treated with MG132, CLN5 expression was not detectable by western blotting. Data from transduced cultures are currently inconclusive. These results suggest that in this cell population, CLN5 mRNA is unaltered in CLN6 NCL. These methods may subsequently be translated to primary cultures as a foundation for on-going investigations into NCL protein interactions in ovine cultures.

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  • Multiple Dosing of RHDV VLP to Enhance the Anti-tumour Response

    Sadrolodabai, Yasmin (2014)

    Undergraduate thesis
    University of Otago

    Metastatic melanoma has a poor prognosis, with a median survival of 13 months after diagnosis. New Zealand has among the highest melanoma rates in the world with more than 2000 cases registered every year. Metastatic melanoma continues to be a challenging disease to treat, but recent immunotherapeutic approaches have demonstrated promising results. Our laboratory has developed a cancer vaccine using a virus-like particle (VLP) derived from Rabbit hemorrhagic disease virus (RHDV), which acts as a highly immunogenic scaffold to deliver tumour-associated antigens (TAAs) to the immune system. In vivo studies to date have shown that one or two doses of the VLP carrying gp100 (a melanoma-associated antigen) can specifically activate an anti-tumour response and trigger the formation of immunological memory against gp100 to prevent tumour recurrence. Administering multiple doses of a vaccine often achieves better responses in vivo, so the key aim of this study was to determine what effect multiple dosing of RHDV VLP coupled to gp100 would have on the anti-tumour response. RHDV VLP was successfully synthesized in a baculovirus expression system using Sf9 insect cells and subsequently used in a series of in vivo experiments. C57BL/6 mice were used in all experiments, receiving either 1, 3 or 6 doses of gp100-carrying VLP (n = 5 per group). An in vivo cytotoxicity assay showed that 3 doses of the VLP vaccine given 5 days apart elicited the highest levels of antigen-specific lysis in a target cell population, compared to a single dose or controls. Therapeutic tumour trials also showed that multiple dosing elicited a stronger anti-tumour response – mice that were first inoculated with B16 melanoma cells and then received 3 or 6 doses of the vaccine 5 days apart had the best overall survival, compared to controls and those that received a single dose. Mice that were tumour-free for 50 days were then rechallenged with B16 cells to assess the immunological memory response, and were found to have increased overall survival, with one mouse from the 3 dose cohort remaining tumour-free. The antibody response against the VLP in these mice was also examined via indirect ELISA. It was found that high levels of antibody against the capsid protein of the VLP were produced in all treated mice, which increased with each additional dose of the vaccine administered. A VLP uptake assay identified that the presence of antibody against the VLP can enhance the early uptake of VLP by DCs, but whether this has an effect on the anti-tumour response remains unclear. Overall, these preliminary results show that treatment involving multiple dosing of RHDV VLP coupled to the melanoma-associated antigen gp100 does somewhat enhance the anti-tumour response in vivo compared to treatment with a single dose, but the reasons for this need to be investigated further. Future work will focus on determining the role that the antibodies against the VLP play in the anti-tumour response, especially in relation to antigen-presenting cells, and further optimizing the vaccine for clinical trials.

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  • The involvement of mGluR1 hyper-activation in the progression of cerebellar ataxia in SCA1 mice

    Desai, Heena Nitin (2014)

    Undergraduate thesis
    University of Otago

    Spinocerebellar ataxia type 1 (SCA1) is an incurable, autosomal dominant progressive neurodegenerative disorder characterised by ataxia, progressive motor deterioration and selective neuronal loss in the cerebellum. It results from a CAG trinucleotide repeat expansion within the SCA1 gene product, ataxin-1. Metabotropic glutamate receptors type 1 (mGluR1) mobilise calcium from intracellular stores as part of their key role in cerebellar synaptic plasticity and motor learning and may be involved in the progression of ataxic symptoms. In this study, we use an 82Q transgenic mouse model of SCA1 where the CAG expansion is restricted to mouse cerebellar Purkinje neurons (PNs), the primary site of SCA1 pathology. This restricted expression in the PNs is achieved by tetracycline-controlled system. We use doxycycline to repress transgene expression at early (6 weeks) and mid (12 weeks) stages of the disease. Our aim is to use this model to identify potential mechanisms that contribute to the early stages of the progression of SCA1. We hypothesise that changes in mGluR1 expression underlie the progression from early pre-symptomatic to ataxia symptoms. Behavioural testing involved using an accelerating rotarod apparatus to assess motor performance and learning. 6 week old SCA1 transgenic mice exhibited mild ataxic motor symptoms (P < 0.01, two way ANOVA, n = 12) that progressed further at 12 weeks of age (P < 0.05, two way ANOVA, n = 7). Doxycycline treatment to repress the transgene expression prevented the mild ataxic symptoms at 6 weeks and reversed the progressively worse ataxic symptoms at 12 weeks of age. Immunohistochemistry experiments showed an increase in mGluR1 expression specifically in the molecular layer of 12 week old SCA1 mice (P < 0.05, two way ANOVA, n = 4). Doxycycline treatment did not prevent this enhanced expression of mGluR1, suggesting that enhanced mGluR1 expression may precede the onset of behavioural ataxia. Cell attached patch clamp recordings from PNs in SCA1 transgenic mice showed a decrease in instantaneous action potential (spike) firing frequency in comparison to PNs from FVB mice (P < 0.01, unpaired t-test with Welch’s correction, FVB: n = 3, SCA: n = 10). The application of Picrotoxin (PTX), a GABAA receptor antagonist resulted in: a non-significant trend towards an increase in instantaneous frequency and decrease in instantaneous firing irregularity of PNs from SCA1 mice. These data suggest a more powerful inhibitory influence in the cerebellar cortex of SCA1 mice compared with FVB mice. Overall, these results suggest that enhanced mGluR1 expression may disrupt PN calcium homeostasis leading to changes in PN firing and cerebellar output that drives the progression of SCA1. Our findings have important implications for the treatment of this rare but incurable human ataxia. The mGluR1 may be a potential therapeutic target for treating patients that are mildly symptomatic in the early stages of the disease.

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  • Phenotyping Langerhans cell like cell treated with microparticles from keratinocytes expressing human papilloma viruse16 E7 oncoprotein

    Zhang , Junda (2014)

    Undergraduate thesis
    University of Otago

    Cervical cancer in females is a worldwide health issue. High risk subtype of human papilloma viruses (HPV) are involved as a major risk factor. HPV oncogenes, E7 and E6 which are over-expressed in the host cells and promote malignant transformation. There is also evidence of HPVE7 involvement in immune modulation. Microparticles (MP), a type of small membrane fragments (0.1um ~1um) released by activated cells, have been implicated in suppressing immunity following interaction with antigen presenting cells. Our laboratory has previously reported up-regulation of microparticle secretion by HPV16E7 expressing keratinocytes. A component of this study is to advancing the understanding of the effect of keratinocyte microparticle on the phenotype of langerhans cells. Increasingly, roles for p53 in regulation of the immune response is being recognized. In this project, the effect of p53 status in langerhans cells on maintianing the immune phenotype will also be tested. The HPV16E7 oncoprotein expressing mouse keratinocyte (E7-PDV) cell line was established following lentiviral transduction, for microparticle (MP) production. Wild-type (p53+) Langerhans cell-like cells (LCLC) and p53 deficient (p53-) LCLC, which were generated from murine bone marrow cells resembling the phenotype of epidermal LC, were used in this study. The effect of MP on LCLC phenotype (CD40, CD86, E-cadherin and cytokine production) was investigated following 48 h co-culture. Compared to the control PDV cell lines, HPV16E7 expressing PDV were found to produce more MP, suggesting a poteintial role for oncoprotein HPV16E7 in inducing MP production. In response to lipopolysacharride stimulation, the up-regulation of inflammatory surface marker CD40 on p53- LCLC was abrogated compared to wild-type LCLC and E-cadherin expression was also found to be low compare to that of wild-type LCLC. This suggests, to some extend, a potential role for p53 protein in maintaining the proper immune phenotype of normal LCLCs. Moreover, comparing to control groups, the same amount of MP from E7-PDV found to have an inhibitory effect on CD40 expression and it also reduced inflammatory cytokine interleukin 12 productions in LPS-stimulated LCLC. The altered LCLC phenotypes subject to MP treatment had confirmed the hypothesis that HPV16E7 induced MP had a modulating effect on the phenotype of LCLC. Finally, the combined down-regulating effect on CD40 expression was observed when MP treatment was applied to p53- LCLC suggested that the MP effect on CD40 expression was p53 independent. The findings of phenotypic alteration of LCLC subject to MP treatment has unveiled the potential role of HPV-induced MP in immune supression that might provide a mechanism to contribute HPV persistence in the skin.  

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  • Structure, stratigraphy and metamorphism in the upper Hakataramea valley area, South Canterbury, New Zealand

    Fagan, Robert Keith (1971)

    Undergraduate thesis
    University of Otago

    vi, 73 leaves ; 30 cm. Includes bibliographical references. University of Otago department: Geology.

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  • Lhx9 is required for urogenital ridge development and ovarian function

    Workman, Stephanie (2017)

    Undergraduate thesis
    University of Otago

    While there has been extensive research into the differentiation of sexually dimorphic gonads, there is much to learn about the bi-potential structure they arise from, the urogenital ridge (UGR). This gap in knowledge is imperative in the contexts of disorders of sex development and infertility, of which many cases have unknown aetiology. The transcription factor LIM Homeobox 9 (Lhx9) has been shown to have a functional role in the development of the UGR. Lhx9 -/- mice display gonadal agenesis and complete male to female sex reversal. Little is known about the regulation of Lhx9 gene expression in the UGR or its role in the greater genetic networks of reproductive development and beyond. We hypothesised that Lhx9 expression is regulated by Notch signalling in the UGR. To investigate the regulation of Lhx9 in the UGR in situ hybridisation was used to analyse the expression patterns of Lhx9, Notch (receptor), and Hes1 (Notch downstream effector) in the embryonic mouse gonad. Overlapping expression patterns in the UGR suggested a coregulatory interaction between the genes. This was further demonstrated by explant culture of embryonic gonads in the presence of the Notch pathway inhibitor DAPT. RT-qPCR revealed reduced Lhx9 expression in RNA extracted from the treated gonads, providing a strong case for a regulatory relationship between Notch and Lhx9. Due to declines in Lhx9 heterozygote (Lhx9+/-) fertility and embryo viability we hypothesised that a reduction in Lhx9 expression would result in impaired fertility in the mouse model. RNA extracted from the gonads of Lhx9+/- embryos was used for RT-qPCR to determine the relative expression of markers of key cell types in the developing gonad. Significant changes in the expression of markers of both male and female somatic and germ cells were found. This raised the question of whether Lhx9 was expressed in the adult ovary, and if so were the observed fertility declines due to reduced Lhx9 expression. In situ hybridisation revealed the novel discovery of Lhx9 expression localised to the follicles of the ovary, this was confirmed by immunohistochemistry. RT-qPCR of heterozygote ovaries revealed trends of reduced expression of critical ovarian fertility genes, a finding reflected in abnormalities seen in histological analysis. These results provide significant evidence for the role of Lhx9 in UGR development and the adult ovary, and offer direction for further investigation into its potential role in the underlying genetic networks DSD and infertility.

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  • Identification of Transporters Involved in Drug-drug Interactions During Gout Treatment in Primary Rat Hepatocytes

    Nguyen, Khanh Ho Kim (2017)

    Undergraduate thesis
    University of Otago

    Gout is one of the most common form of inflammatory arthritis, with hyperuricemia as the major risk factor. Chronic hyperuricemia, or high level of serum uric acid (SUA), can lead to the formation of monosodium urate crystals (MSU) in the joints, which can result in acute flares in gout. Allopurinol is the gold standard therapy for gout, with its active metabolite, oxypurinol, acts to inhibit the enzyme responsible for uric acid synthesis, xanthine oxidase (XO), thus lowering SUA level. Moreover, ~ 70% of US adults with gout also has hypertension, which is often treated with diuretics such as furosemide. However, concomitant treatment with furosemide compromises the therapeutic effects of allopurinol. The molecular mechanisms underlying this adverse drug-drug interaction are unknown. Based on current knowledge of the transport of furosemide and allopurinol/oxypurinol by transporters in the kidney, and the fact that similar transporter setups exists in the liver, as well as evidence from clinical studies, we hypothesise that transporters known to translocate these drugs in the kidney are responsible for the drug-drug interactions in the liver, where allopurinol/oxypurinol act to lower SUA. Hence, the aim of this project was to mimic the in vivo situation in gout patients with our cell model by treating cultured primary rat hepatocytes with gout-associated drugs. The functional outputs were assessed by measuring extra- (EUA) and intracellular uric acid (IUA) level. First, we were able to successfully establish a protocol to extract primary hepatocytes via in situ perfusion method. These extracted cells had polygonal morphology, characteristic of primary hepatocytes described in the literature. We characterised the gene expression profile of urate transporters and its converting enzymes in these hepatocytes and in the rat liver tissue. In both the extracted hepatocytes (n=3) and the tissue (n=6), qPCR analysis confirmed expression of the following genes: AOX1, XO, Oat2, Oat3, Glut9, Mrp4, Npt1, Npt4, and Abcg2. Furthermore, immunoblotting was carried out to confirm protein expression of our main proteins of interest: XO, Oat2, Glut9, Mrp4, and Abcg2. However, a temporal profile of the gene expression of the hepatocytes found that, after 48h there was a downregulation of most of the genes, except for Npt4 and Mrp4, which seemed to have not changed, and Abcg2 seemed to be upregulated (n = 1). From functional studies, we treated the cells with combinations of 250 μM allopurinol, 250 μM oxypurinol, 1 mM furosemide, and 1 mM probenecid. 24h after treatment, the cells and media were harvested for analysis. Our results showed that there was a significant decrease in EUA in cells treated with probenecid, which was in agreement with our model. However, due to low sample numbers, we were not able to draw any conclusion from these functional results. Additionally, a knockdown study was performed to evaluate the contribution of Oat2 and Glut9 to the transport of allopurinol and oxypurinol. Results from one set of experiment suggest that Oat2 might play a bigger role in transport of these drugs than Glut9. Further experiments are required to confirm transport of allopurinol/oxypurinol by these two transporters.

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  • Stratigraphy, tectonics, and provenance of rocks in the Wether Hill area, Western Southland

    Willsman, Andrew (1991)

    Undergraduate thesis
    University of Otago

    Digital copy stored under Section 55 of the NZ Copyright Act.

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  • The geology of the Eastern Saddle Road area, Manawatu, N.I., N.Z.

    Grammer, Terrence Ronald (1971)

    Undergraduate thesis
    University of Otago

    Digital copy stored under Section 55 of the NZ Copyright Act.

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  • Modulation of DNA methylation by L-ascorbate and 5-aza-2’-deoxycytidine in murine embryonic stem cells

    Bridgman, Luke David (2017)

    Undergraduate thesis
    University of Otago

    Cytosine methylation, normally found on cytosine residues adjacent to guanine (i.e., a CpG dinucleotide), is one means by which long-term gene repression occurs. Immediately after semi-conservative DNA replication, CpG dinucleotides on the replicated daughter strand are unmethylated, giving the “hemimethylated” state, where one DNA strand is methylated and one unmethylated. Hemimethylation is usually corrected through complementary methylation by Dnmt1 maintenance methyltransferase, but oxidative stress can inhibit Dnmt1, and so replicated DNA will remain hemimethylated. The Morison laboratory had new evidence that this aberrant hemimethylated DNA is “corrected” by the Tet family of enzymes, which actively catalyse the conversion of parent strand 5-mC to 5-carboxylcytosine (5-cC), that is subsequently replaced with cytosine by base excision repair. This study developed a murine embryonic stem cell model through which the molecular basis of active demethylation could be investigated. It was hypothesised that Tet family enzymes actively demethylate DNA, following oxidative stress-mediated inhibition of Dnmt1; i.e., after the generation of hemimethylated DNA. To model oxidative stress-induced hemimethylation the Dnmt1 inhibitor decitabine was used. The effect of ascorbate on Tet activity, both alone and in conjunction with decitabine was also assessed. Ascorbate increases Tet activity by increasing regeneration of Fe2+, whilst decitabine (5-aza-2’-deoxycytidine) inhibits Dnmt1 by binding and sequestering it. We hypothesise that when Tet is activated using ascorbate and Dnmt1 is inhibited by decitabine the proportion of unmethylated DNA should increase, due to the creation of hemimethylated DNA by decitabine and the upregulation of Tet activity by ascorbate. To perform this research, murine embryonic stem (ES) cells were maintained and manipulated in cell culture. Cell lines were synchronised to G1 by thymidine block. ES cells received differential treatments: control culture, + decitabine, + ascorbate, and + decitabine / + ascorbate. Samples were extracted at 2, 4 and 6 hours post-release from thymidine synchronisation. Hairpin linkers were used to maintain the connection between complementary DNA strands throughout PCR amplification, allowing comparison of parent-daughter strand methylation to identify hemimethylated sequences. Hairpin linkers were synthesised for three highly methylated genes: Asz1, Ckt2 and Kcnv2. Two next-generation sequencing libraries were prepared, containing 144 and 288 samples respectively, and sequenced using the high-throughput Illumina MiSeq platform. Bisulfite-converted sequences were aligned using BiQ Analyzer HT software, and methylation symmetry in complementary DNA strands determined using RStudio. Methylation proportions were averaged and/or correlated with results for each replicate, and plotted. This project identified that ascorbate induced marked demethylation in ES cells, whilst decitabine caused large increases in hemimethylation, but no increase in demethylation. When decitabine was added in conjunction with ascorbate, increasing demethylation was observed. These findings demonstrate that decitabine has the potential to induce marked hemimethylation, even in wild-type cells, in ascorbate-deficient culture. There was also evidence to suggest that in the presence of hemimethylated target sequence, ascorbate is necessary to allow Tet activity. This was seen in Tet-Triple knockout ES cells, where ascorbate failed to induce demethylation. Overall, the results support the hypothesis that hemimethylated DNA has the potential to induce Tet enzyme activity.

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  • Novel Organic Carbon Monoxide Releasing Molecules as a Potential Treatment for Triple Negative Breast Cancer

    Gunatunga, Kishan (2011)

    Undergraduate thesis
    University of Otago

    Carbon monoxide (CO) plays a role in many physiological and pathophysiological processes as a second messenger. Emerging evidence reveals the potential CO has as a therapeutic agent as it has been implicated in the modulation in a range of intracellular functions including apoptosis and proliferation. In the case of cancer, specifically triple negative breast cancer (TNBC), there is very little information regarding the effects of this molecule. Here we hypothesize that the targeted delivery of CO to a tumour will result in an anti-cancer effect in TNBC. The current study examines a novel class of compounds termed organic CO releasing molecules (CORMs) (CO-1 – CO-8) and previously published metal containing CORMs (CORM-2), as potential treatments for TNBC. Firstly a wide range of synthesised novel organic CORMs were screened for toxicity in MDA-MB-231 cells, a model for TNBC, and the lead compound CO-1 was identified from a range of 8 potential candidates (CO-1 – CO-8). Analysis of cell viability data revealed that CO-1 (1 – 200 μM) resulted in significant reductions in cell viability with an IC75 value of around 5 μM in the MDA-MB-231 TNBC cell line, while the by-product of CO-1, BP-1, demonstrated no residual cytotoxic effects. Time course and gas chromatograph-mass spectrometry (GC-MS) studies revealed that the compound released CO at a slow rate with a half-life in vitro between 9 and 24 hours. The ability of CO-1 and CORM-2 to modulate cell death via the induction of apoptosis was demonstrated using Annexin V conjugated to fluorescein (FITC) and propidium iodide (PI) staining followed by FACS analysis. CO-1 was able to induce apoptosis in MDA-MB-231 cells at both low (10 μM) and high (200 μM) concentrations (6% and 6% respectively) with no apoptotic or necrotic effects being observed when cells were treated with the by-product of CO-1, BP-1. The transition metal containing CORM-2 (200 μM) did not increase apoptotic markers compared to control, however treatment of cells with its “inactive” counterpart iCORM-2 (200 μM) resulted in a significant increase (7%) in apoptosis. In addition high (200 μM) but not low (5 and 10 μM) concentrations of CO-1 and CORM-2 produced a significant increase in the percentage of cells with a damaged mitochondrial membrane (3% and 5% for CO-1 and CORM-2 respectively), indicating that CO may have some concentration specific effects in vitro. High (200 μM) concentrations of both CO-1 and CORM-2 were also shown to induce mitochondrial damage in the MDA-MB-231 cell line and further to the potential anti-cancer effects of the novel compound CO-1, we have shown that low (10 μM) concentrations of the molecule causes a 1.2-fold and 1.4-fold increase in caspase 3 and p53 expression and a 1.2-fold increase in caspase 3 activation. The safety of both organic and transition metal CORMs were also assessed in the renal epithelial MDCK cell line. In MDCK cells treated with CO-1 (10 and 200 μM), COM-2 and iCORM-2 (20 and 100 μM) showed histopathological changes indicative of cell death were observed. These changes were not present in cells treated with the by-product of CO-1, BP-1. Interestingly the changes in histological architecture in MDCK cells treated with iCORM-2 appeared more extensive and severe that in cells treated with the active form of the compound CORM-2. Furthermore treatment of MDCK cells with low (10 μM) concentrations of CO-1, 20 and 200 μM CORM-2 and 200 μM iCORM-2 resulted in G2/M cell cycle arrest in the MDCK cell line. The current study proved CO-1, to be a safe and efficacious pharmacological agent with the ability to induce a cytotoxic and cytostatic effect in the MDA-MB-231 and MDCK cell line with no residual toxic effects resulting from treatment of cell with the by-product of CO-1 (BP-1). Our findings cast doubt over the notion that existing transition metal CORMs in their “inactive” form are not without biological effects. Therefore the current study has shown that novel organic CORMs have a combination of properties that translate into a desirable and potential treatment for TNBC.

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  • Investigating the role of peroxiredoxin in hydrogen peroxide signalling

    Carrad, Rose Jane (2017)

    Undergraduate thesis
    University of Otago

    Hydrogen peroxide (H2O2) was viewed as an unwanted by-product of aerobic respiration for many years but it is now well-established as being involved in many different signalling pathways. H2O2 is reduced to water by peroxiredoxins (Prdx), which make up a family of antioxidant enzymes. Due to high rate constants and the abundancy of Prdx in cells, H2O2 is quickly reduced to water. Therefore, signalling proteins have to compete with Prdx to be oxidised by H2O2. This has resulted in two conflicting theories for how Prdx are involved in H2O2 signalling. The first is the floodgate model, suggesting that Prdx are inactivated by high levels of H2O2, allowing for direct H2O2 oxidation of signalling proteins. The second model, signal peroxidase model, suggests that Prdx are more directly involved in the reversible oxidative activation of specific non-peroxidase thiol-containing proteins. Prdx1, a ubiquitously expressed cytoplasmic enzyme, is one of six isoforms of the Prdx family. Prdx1 has been characterised as acting in a redox relay mechanism in the H2O2 activation of the apoptosis signal-regulating kinase 1 (ASK1) apoptotic pathway. ASK1 activates apoptosis via the phosphorylation and activation of p38. This set of experiments aims to characterise the role of Prdx1 in H2O2 signal transduction, looking specifically at H2O2 induced apoptosis via the phosphorylation and activation of p38. The model systems used for this set of experiments were Hap1 wild type and Prdx1 knockout cells. Experiments carried out to measure the effect that Prdx1 knockout has on cell growth were done through the observation of Prdx1 knockout and WT growth rates. There was no observed difference in growth rates providing evidence that Prdx1 knockout does not affect cell growth. Secondly the effect that Prdx1 has on H2O2 induced phosphorylation and activation of p38 was determined through the comparison of p-p38 in WT and Prdx1 knockout cell lines that had been treated with H2O2. Due to nonspecific binding of the anti-phoshpo-p38 antibody these experiments remain inconclusive. Therefore, H2O2 induced activation of apoptosis was measured in Prdx1 knockout and WT cell lines by caspase-3 activity. Again, there was no observed difference of H2O2 induced apoptosis between the Prdx1 knockout and WT cell lines. This result gives evidence that Prdx1 does not have a critical role in the H2O2 signalling cascade that activates apoptosis. Further work to supplement these experiments is to observe H2O2 induced phosphorylation of a broad range of human kinases between Prdx1 knockout and WT cell lines. There were various kinases that were seen to be differentially phosphorylated in Prdx1 knockout cells. C-Jun, Hck, HSP60, PYK2, STAT2, STAT5a and STAT5a/b were seen to be differentially phosphorylated in Prdx1 knockout cells when treated with H2O2. In conclusion, this set of experiments gives evidence that Prdx1 does not have a crucial role in cell growth or H2O2 induced apoptosis in Hap1 cells. Therefore, these results do not support either theory for how Prdx are involved in H2O2 signalling in the cell. Preliminary phospho-kinase array results show a possible role of Prdx1 in signal transduction as per the signal peroxidase model.

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  • Functional Role of Uric Acid in Cardiac Stem Cell Homeostasis

    Cheakhun, Cindy (2016)

    Undergraduate thesis
    University of Otago

    In New Zealand, cardiovascular disease (CVD) affects 1 in 20 adults and is the leading cause of mortality. Cardiac pathophysiology along with diabetes mellitus is historically associated with glucose metabolism, but recently it has been suggested that elevated serum uric acid (SUA) could be a major driver of these complications. Cardiac stem cells (CSCs) are a new therapy approach for CVD, however, the therapeutic effects are usually mixed. A major contributing factor could be the necrotic environment that the stem cells are being isolated from or transplanted back into. Elevated SUA could contribute significantly to the necrotic environment. Hence, our goal was to characterise the effects of elevated SUA, reflected by changes in extracellular uric acid (EUA) on cardiac progenitor cell (CPC) function in order to improve stem cell therapy, as CPCs are also investigated for use in stem cell therapy and are similar to CSC in terms of physiology and function. We characterised the uric acid (UA) transporter expression profile in human CPCs including known renal transporters: ABCG2, MRP4, GLUT9, URAT1, OAT1, OAT2, OAT4 and NPT4, using qPCR. Human CPCs as well as mRNA from human heart tissue express UA efflux transporters ABCG2 and MRP4 to a greater extent than UA influx transporter GLUT9, irrespective of diabetic status. The expression of GLUT9, ABCG2 and MRP4 were also confirmed at the protein level through western blot analyses of proteins extracted from CPCs. This transporter profile in human CPCs support our new concept of a ‘cellular uric acid homeostasis’ (CUAH), and the fact that a disturbance of CUAH, as in diabetes mellitus, may be detrimental to the function of CPCs and ultimately affect stem cell therapy. From functional assays, it appears that CPCs are sensitive to changes in EUA concentrations. By the end of UA treatment, 300μM UA treatment, which is considered normal SUA, had the most viable cells. The amount of viable cells decreased with 200μM UA and 500μM UA significantly (p<0.01). In sum, the results support the idea that CUAH is crucial for the functionality of CPCs, and therefore changes in CUAH, or the environment that stem cells and progenitor cells are transplanted into can impair their ability to integrate into the host tissue, and therefore decrease the efficacy of stem cell therapy.

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  • Sex-differential expression of microRNAs during mammalian neurodevelopment

    Szakats, Susanna Katharine (2016)

    Undergraduate thesis
    University of Otago

    Pervasive sex differences between male and female brain development have been implicated in neurodevelopmental disorders featuring a distinct sex bias, such as autism. To further our understanding of sex differences we hypothesised that sex-differential microRNA expression occurs in the developing brain. MicroRNAs are powerful regulators of gene expression whose function is essential for normal brain development, and are thought to mediate development of sex differences. Small RNA sequencing was performed with RNA isolated from E15.5 mouse brains (n=3 per sex), representing early brain differentiation. Comparison of microRNA expression identified 12 microRNAs that were differentially expressed between sexes (p<0.05). Differences in expression were validated using real time quantitative RT-qPCR for miR-10, and miR-205 and miR-5099. miR-10 is encoded within the Hox gene complex, a cluster of genes conferring positional identity to structures along the anterior-posterior axis of the developing embryo. As miR-10 is thought to be controlled in the same spatially co-linear manner as the Hox genes, we anticipated that it has a distinctive expression domain. Using in situ hybridisation with a locked nucleic acid (LNA)-probe targeting miR-10, strong staining for miR-10 expression was found in the hindbrain and anterior spinal cord of embryos at E15.5. Not only did we investigate known microRNAs, we also analysed novel microRNAs aligning to the mouse Anti-Müllerian Hormone AMH gene. Previous experiments identified a non-coding RNA (ncRNA) transcribed at this locus, bearing hallmarks of a primary microRNA. Subsequent preliminary small RNA sequencing at E12.5 (n=1 per sex) showed microRNA profiles at the AMH locus unique to each sex. Using RT-qPCR validation, female-specific expression of a novel microRNA aligning to exon 3 of AMH was determined. Interestingly, an increase in microRNA expression occurred in mouse lines where the exons 1 and 2 of the AMH gene were deleted. This observation lead to identification of a putative repressor element within exon 2, thought to negatively regulate expression of ncAMH. Our findings indicate that sex-differential expression of microRNAs occurs in the developing mouse brain during a key stage of brain patterning. The identified microRNAs are known to influence development and neurological functions, and therefore their expression in a sex-differential manner may contribute to sex differences established in the developing brain.

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  • Efficacy of Prototype Vaccines for Prevention of Tuberculosis

    Sandford, Sarah (2016)

    Undergraduate thesis
    University of Otago

    Tuberculosis (TB) is a pulmonary disease caused by Mycobacterium tuberculosis, which killed 1.5 million people in 2014. The current TB vaccine, live attenuated Mycobacterium bovis bacille Calmette Guérin (BCG), effectively protects against neonatal TB yet has variable efficacy against pulmonary TB in adults. Consequently, a more effective preventive vaccine is needed. This project aimed to characterise the ability of two innovative new vaccines to improve antigen delivery and induce protective mucosal immune responses against mycobacterial infection in mice. The first vaccine approach employed sustained release of protein antigen together with adjuvants to enhance immunogenicity. To that end, a vaccine comprising the mycobacterial antigen 85B (Ag85B) with poly lactic-co-glycolic acid nanoparticles in a thermoresponsive chitosan gel was developed. This vaccine was hypothesised to prolong antigen exposure, enhancing the development of an effective immune response, and reducing the need for booster vaccinations. The second approach was designed to activate mucosal immune responses, using non-pathogenic Lactococcus lactis as a vector. Genetically engineered to overexpress the model antigen Ovalbumin (Ova) on its surface via a Group A Streptococcus serotype M1 Pilus (L. lactis PilM1-Ova), this prototype vaccine was hypothesised to improve antigen delivery to the mucosal immune system. The chitosan gel vaccine containing Ag85B, and the L. lactis PilM1-Ova vaccine were administered separately to groups of mice. For both vaccines, the activation state and cytokine responses of antigen-specific T cell populations were detected using flow cytometry. Compared to BCG, the chitosan gel vaccine did not induce higher frequencies of activated, proliferating, cytokine-producing, or memory Ag85B-specific T cells, and failed to protect against an intranasal BCG challenge. By contrast, L. lactis PilM1-Ova induced more activated, proliferating, cytokine-producing and memory Ova-specific T cells than recombinant BCG expressing Ova (BCG-Ova). Although L. lactis PilM1-Ova was not protective against intranasal BCG-Ova challenge, this may have been due to slow in vivo growth of the recombinant BCG-Ova. This work suggests that the chitosan gel vaccine requires further development to improve immunogenicity, for example by selecting a different antigen or antigen dose. L. lactis PilM1 shows promise as a vaccine delivery system for TB and is worthy of further studies, ideally with expression of an endogenous mycobacterial antigen to measure vaccine-induced antigen-specific T cell responses and protection against TB.

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  • Investigating the anti-invasive properties of dexamethasone in Fn14 positive triple negative breast cancer.

    Harris, Joshua (2016)

    Undergraduate thesis
    University of Otago

    Triple-negative breast cancer (TNBC) is an aggressive disease subtype with no current targeted systemic therapies due to a lack of actionable molecular targets. Efforts to identify therapeutic targets in TNBC have revealed that Fn14, a cytokine receptor, is over-expressed in most invasive breast cancers (1, 2) but not in healthy breast tissue. Fn14 has been shown to activate canonical NF-κB signalling (3), which in turn, transactivates the Fn14 promoter (3) leading to an autoregulatory loop that may drive invasive capacity (2) in breast cancer. We hypothesised that repression of the Fn14/NF-κB feedback loop may reduce Fn14 expression and invasive capacity in breast cancer. As corticosteroids are known to attenuate NF-κB activity through glucocorticoid receptor alpha (GR⍺), we postulated that corticosteroids might impact Fn14 expression. We investigated this likelihood in three TNBC cell lines positive for GR⍺ and Fn14 (BT-549, MDA-MB-436 and SUM149PT). Following 24h treatment with the corticosteroid, dexamethasone (DEX), we observed a decrease in Fn14 protein expression. In parallel, DEX reduced invasive capacity in vitro by an average of 40% with the most significant and consistent response in BT-549 cells. The DEX-mediated reduction in Fn14 expression and invasive capacity are likely due to a repressive relationship between GR⍺ and NF-κB. However, the potential mechanisms of repression are complex and poorly understood in breast cancer. To investigate this mechanism, we first used immunocytochemistry to visualise the spatial location of GR⍺ and NF-κB (p65) in BT-549 cells after 24h of DEX. Our results showed rapid nuclear translocation of GR⍺ and subtle nuclear exclusion of p65. We quantified this observation using western blotting over a 24h time course with DEX using subcellular fractionation of BT-549 cells. These data suggest that a repressive physical interaction between GR⍺ and p65 is unlikely, due to the opposite movement of transcription factors between cellular compartments. Interestingly, IκB⍺, an inhibitor of NF-κB, exhibited a subtle DEX-mediated increase in protein expression over 24h, corresponding with the cytoplasmic retention of p65. Our data, therefore, supports a model that repression of NF-κB signalling may be achieved through de novo NF-κB inhibition. Additionally, BT-549 cells transfected with an NF-κB-response element/luciferase reporter plasmid showed an intriguing increase in luciferase activity following 24h of DEX. It was later shown that this may have been caused by DEX-induced glucocorticoid resistance due to the downregulation of GR⍺ after DEX treatment. These results demonstrate a novel mechanism for Fn14-mediated invasion. However, investigation in additional cell lines is required. The induction of DEX-mediated glucocorticoid resistance also questions the clinical significance of glucocorticoid based therapy in the treatment of TNBC.

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  • Anatomical Characterisation of Prolactin Receptor Expression in the Developing Mouse Brain

    Boyes, Kendra Michelle (2016)

    Undergraduate thesis
    University of Otago

    Prolactin signalling through the prolactin receptor plays a significant role in many physiological processes. The diverse biological functions attributed to prolactin are largely dependent on the distribution of its receptor. In particular, the distribution of the prolactin receptor has been well characterised in the adult brain of rodents, but has yet to be fully documented in the developing brain of neonates. To determine the role of the prolactin receptor in the developing brain of neonates, we have developed a transgenic mouse in which cre-recombinase is expressed under the control of an internal ribosomal entry site (IRES) in the long form of the prolactin receptor gene (PrlrL-IRES-Cre). The PrlrL-IRES-Cre mouse has been crossed with a cre-dependent tau-GFP reporter line. In this study tau-GFP is expressed wherever the prolactin receptor gene has been transcribed. The tau-GFP associates with microtubules in neuronal processes, which is important, because in addition to identifying neuronal cell bodies expressing the prolactin receptor, we were able to study the projections of prolactin-responsive neurons for the first time. Post natal day (PND) 1 (n = 8), PND 14 (n = 8) and PND 28 (n = 6) male and female mouse brains were perfused using 4% paraformaldahyde, cut in 20 micron serial sections and thaw mounted onto slides. To visualise PrlrL expressing cells, immunohistochemistry was performed. Sections were incubated with rabbit polyclonal anti-GFP (green fluorescent protein) (A-6455, Life Technologies), for 48 hours at 4℃, followed by an incubation with goat anti-rabbit biotinylated secondary antibody (BA-1000, Vector) for 3 hours. Labelled cells were visualised using nickle enhanced 3,3'-diaminobenzidine (DAB), and identified using an Olympus AX70 light microscope. Staining showed minimal expression in any part of the brain of PND 1 mice, whereas staining could be identified in the choroid plexus, anterior ventral periventricular nucleus and arcuate nucleus of PND 14 and 28 mice. Interestingly, minimal staining was observed in the amygdala and no staining was observed in the paraventricular nucleus in any of the ages examined. These are areas that exhibit high levels of PrlrL expression in adult mice. The increase in PrlrL expression may indicate an age dependent upregulation of the prolactin receptor through the availability of the PrlrL gene, likely driven by hormonal changes associated with puberty. The age-associated increase in PrlrL expression in the anteroventral periventricular nucleus and arcuate nucleus in prepubertal animals suggests a developmental role for prolactin in these regions.

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  • Does prolactin act directly on AgRP neurons in the arcuate nucleus?

    MacLeod, Morgan Anna (2016)

    Undergraduate thesis
    University of Otago

    The development of a positive energy balance occurs during pregnancy to support the growth of fetal and maternal tissues, and to increase energy stores in preparation for the metabolic demands of lactation. Leptin levels increase as pregnancy advances, along with appetite and fat deposition. This is unusual, as in the non-pregnant state, leptin decreases food intake and increases energy mobilization. Therefore, leptin is not being recognized during pregnancy. Leptin-responsive neurons, such as Agouti-related peptide (AgRP) and Pro-opiomelanocortin (POMC) neurons in the arcuate nucleus of the hypothalamus, do not respond normally to leptin during pregnancy. It is likely that changes in gestational hormonal profile have a role in mediating leptin resistance. Increased plasma prolactin is observed during pregnancy and, in addition, there are prolactin receptors in the same regions of the brain that control food intake. Prolactin is also known to have an orexigenic effect. We hypothesise that prolactin is a mediator of this increased appetite and resistance to leptin during pregnancy. The aim of this study was to examine whether prolactin directly targets leptin-responsive neurons in the arcuate nucleus. We examined the AgRP neurons, known to be involved in stimulating food intake. Transgenic mouse models and double-label immunohistochemistry were used to look at whether prolactin action is observed in neurons that express AgRP. In separate experiments, AgRP-Cre-tdTomato mice and AgRP-Cre-tauGFP mice were injected with prolactin and perfused. The brains were used to detect prolactin-induced phosphorylation of STAT5, an indicator of prolactin action. The sections were then double-labelled to identify AgRP neurons with labeling of either the tdTomato transgene or GFP respectively. AgRP neurons were readily detected in the arcuate nucleus. Many pSTAT5-stained nuclei were also detected in the arcuate nucleus of prolactin-treated animals, however, none of the AgRP neurons showed pSTAT5-positive nuclei. To confirm these results, we looked at conditional deletion of the prolactin receptor in AgRP neurons using AgRP-PRLR flox/flox mice. We looked at differences in food intake and body weight between these knockout mice and wildtype littermates, no significant differences were observed. We also used immunohistochemistry to look at GFP expression. These mice would be expected to show Cre-induced expression of GFP if the prolactin receptor was normally expressed in the AgRP neurons. vGAT-Cre-Prlr flox/flox transgenic mice were used as a positive control as previous work suggests that GABA neurons express the prolactin receptor. While GFP was readily detected in the positive controls, no staining of any neurons was visible in the non-pregnant, pregnant or lactating brains from AgRP-PRLR flox/flox mice, indicating that the prolactin receptor gene is not normally expressed in AgRP neurons. From these experiments we were able to conclude that prolactin-induced pSTAT5 does not colocalise with AgRP neurons, and that the prolactin receptor gene is not expressed in AgRP neurons. Therefore, the results obtained did not support our hypothesis that prolactin-induced increases in food intake are mediated by direct actions of prolactin by AgRP cells, and showed that prolactin does not directly act upon AgRP neurons to mediate leptin resistance during pregnancy.

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