Central regulation of food intake and reproduction

The work described in this thesis investigates the actions of hypothalamic neuropeptides involved in the control of energy homeostasis and reproduction. Cerebellin 1 (Cbln1) is a 16-amino acid peptide abundantly expressed in the cerebellum and the hypothalamus. I have shown that Cbln1 increases food intake when administered centrally to rats, and that this effect may be partly mediated by neuropeptide Y. I have also demonstrated that Cbln1 mRNA expression within the ventromedial nucleus of the hypothalamus is up-regulated following a 48-hour fast, suggesting that Cbln1 may have a physiological role in the control of food intake. In addition to hypothalamic centres, the reward system has an important role in the control of feeding. The melanocortin system has a well characterised role in the homeostatic control of food intake. My work suggests that the melanocortin system may also have a previously unknown role within the ventral tegmental area (VTA) in the hedonic control of feeding. Intra- VTA administration of a melanocortin agonist reduced, whereas an antagonist increased, food intake in rats. My work suggests that the VTA melanocortin system may act upstream of dopamine signalling to influence feeding. Kisspeptin is a hypothalamic neuropeptide with a crucial role in reproduction. The hypothalamus contains two populations of kisspeptin neurones, located in the arcuate nucleus (Arc) and in the anteroventral periventricular nucleus. The physiological role of these two populations is unknown. I used recombinant adeno-associated virus (rAAV) to suppress kisspeptin expression within the Arc in female rats. Animals injected bilaterally into the Arc with rAAV-kisspeptin antisense had significantly fewer oestrous cycles, and an increase in oestrous cycle length, compared to controls. In summary, these studies have identified novel roles for Cbln1 and the melanocortin system in the control of food intake, and have demonstrated the importance of Arc kisspeptin signalling in the regulation of reproduction

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Exponential growth, high prevalence of SARS-CoV-2, and vaccine effectiveness associated with the Delta variant

SARS-CoV-2 infections were rising during early summer 2021 in many countries associated with the Delta variant. We assessed RT-PCR swab-positivity in the REal-time Assessment of Community Transmission-1 (REACT-1) study in England. We observed sustained exponential growth with average doubling time (June-July 2021) of 25 days driven by complete replacement of Alpha variant by Delta, and by high prevalence at younger less-vaccinated ages. Unvaccinated people were three times more likely than double-vaccinated people to test positive. However, after adjusting for age and other variables, vaccine effectiveness for double-vaccinated people was estimated at between ~50% and ~60% during this period in England. Increased social mixing in the presence of Delta had the potential to generate sustained growth in infections, even at high levels of vaccination

Hands on: The importance of studio learning in design education

Studio learning and teaching in art and design is still a relatively under-explored zone. While a growing body of literature is beginning to explore what kind of learning and teaching occurs in studio, there is less literature available that explicates why studio learning is important. Three elements highlighting the importance of studio in design learning and teaching are at the centre of this article: the development of artistic/design skills and understanding; the internal and external processes and practices of studio; and how studio models future working environments. This argument has been presented in the context of the authors’ experiences in teaching and supporting a university textile design programme, and has drawn from 2009 student data from a study in the same context. This article has framed such experiences and data in the context of contemporary thinking as expressed in the literature on studio

Central regulation of food intake and reproduction

The work described in this thesis investigates the actions of hypothalamic neuropeptides involved in the control of energy homeostasis and reproduction. Cerebellin 1 (Cbln1) is a 16-amino acid peptide abundantly expressed in the cerebellum and the hypothalamus. I have shown that Cbln1 increases food intake when administered centrally to rats, and that this effect may be partly mediated by neuropeptide Y. I have also demonstrated that Cbln1 mRNA expression within the ventromedial nucleus of the hypothalamus is up-regulated following a 48-hour fast, suggesting that Cbln1 may have a physiological role in the control of food intake. In addition to hypothalamic centres, the reward system has an important role in the control of feeding. The melanocortin system has a well characterised role in the homeostatic control of food intake. My work suggests that the melanocortin system may also have a previously unknown role within the ventral tegmental area (VTA) in the hedonic control of feeding. Intra- VTA administration of a melanocortin agonist reduced, whereas an antagonist increased, food intake in rats. My work suggests that the VTA melanocortin system may act upstream of dopamine signalling to influence feeding. Kisspeptin is a hypothalamic neuropeptide with a crucial role in reproduction. The hypothalamus contains two populations of kisspeptin neurones, located in the arcuate nucleus (Arc) and in the anteroventral periventricular nucleus. The physiological role of these two populations is unknown. I used recombinant adeno-associated virus (rAAV) to suppress kisspeptin expression within the Arc in female rats. Animals injected bilaterally into the Arc with rAAV-kisspeptin antisense had significantly fewer oestrous cycles, and an increase in oestrous cycle length, compared to controls. In summary, these studies have identified novel roles for Cbln1 and the melanocortin system in the control of food intake, and have demonstrated the importance of Arc kisspeptin signalling in the regulation of reproduction.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Quantification of rat kisspeptin using a novel radioimmunoassay

Kisspeptin is a hypothalamic peptide hormone that plays a pivotal role in pubertal onset and reproductive function. Previous studies have examined hypothalamic kisspeptin mRNA expression, either through in situ hybridisation or real-time RT-PCR, as a means quantifying kisspeptin gene expression. However, mRNA expression levels are not always reflected in levels of the translated protein. Kisspeptin-immunoreactivity (IR) has been extensively examined using immunohistochemistry, enabling detection and localisation of kisspeptin perikaya in the arcuate nucleus (ARC) and anteroventral periventricular nucleus (AVPV). However, quantification of kisspeptin-IR remains challenging. We developed a specific rodent radioimmunoassay assay (RIA) capable of detecting and quantifying kisspeptin-IR in rodent tissues. The RIA uses kisspeptin-10 as a standard and radioactive tracer, combined with a commercially available antibody raised to the kisspeptin-10 fragment. Adult female wistar rat brain samples were sectioned at 300 µm and the ARC and AVPV punch micro-dissected. Brain punches were homogenised in extraction buffer and assayed with rodent kisspeptin-RIA. In accord with the pattern of kisspeptin mRNA expression, kisspeptin-IR was detected in both the ARC (47.1±6.2 fmol/punch, mean±SEM n = 15) and AVPV (7.6±1.3 fmol/punch, mean±SEM n = 15). Kisspeptin-IR was also detectable in rat placenta (1.26±0.15 fmol/mg). Reverse phase high pressure liquid chromatography analysis showed that hypothalamic kisspeptin-IR had the same elution profile as a synthetic rodent kisspeptin standard. A specific rodent kisspeptin-RIA will allow accurate quantification of kisspeptin peptide levels within specific tissues in rodent experimental models

Defective monocyte oxidative burst predicts infection in alcoholic hepatitis and is associated with reduced expression of NADPH oxidase

ObjectiveIn order to explain the increased susceptibility to serious infection in alcoholic hepatitis, we evaluated monocyte phagocytosis, aberrations of associated signalling pathways and their reversibility, and whether phagocytic defects could predict subsequent infection.DesignMonocytes were identified from blood samples of 42 patients with severe alcoholic hepatitis using monoclonal antibody to CD14. Phagocytosis and monocyte oxidative burst (MOB) were measured ex vivo using flow cytometry, luminometry and bacterial killing assays. Defects were related to the subsequent development of infection. Intracellular signalling pathways were investigated using western blotting and PCR. Interferon-γ (IFN-γ) was evaluated for its therapeutic potential in reversing phagocytic defects. Paired longitudinal samples were used to evaluate the effect of in vivo prednisolone therapy.ResultsMOB, production of superoxide and bacterial killing in response toEscherichia coliwere markedly impaired in patients with alcoholic hepatitis. Pretreatment MOB predicted development of infection within two weeks with sensitivity and specificity that were superior to available clinical markers. Accordingly, defective MOB was associated with death at 28 and 90 days. Expression of the gp91phoxsubunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was reduced in patients with alcoholic hepatitis demonstrating defective MOB. Monocytes were refractory to IFN-γ stimulation and showed high levels of a negative regulator of cytokine signalling, suppressor of cytokine signalling-1. MOB was unaffected by 7 days in vivo prednisolone therapy.ConclusionsMonocyte oxidative burst and bacterial killing is impaired in alcoholic hepatitis while bacterial uptake by phagocytosis is preserved. Defective MOB is associated with reduced expression of NADPH oxidase in these patients and predicts the development of infection and death.</jats:sec

Quantification of kisspeptin-IR in the rat brain (A) Diagram of a rat brain coronal section showing the position of the punch microdissections for the Hypothalamic anteroventral periventricular nucleus (AVPV) and the arcuate nucleus (ARC).

<p>AVPV at bregma −0.26<b> </b>mm and the ARC at bregma −3.30<b> </b>mm according to the rat brain atlas of Paxinos and Watson. Black circles denote punch positions. Ac, anterior commissure; 3v, third cerebral ventricle. (B) Quantification of kisspeptin-IR in the AVPV and ARC of female rats by RIA. Significantly more kisspeptin-IR was detected in the ARC than in the AVPV. (Mean S.E.M, n = 15<b>–</b>16, ***P<0.001, Student’s t-test).</p

HPLC profile of kisspeptin-immunoreactivity from hypothalamic extracts.

<p>Solid lines- Kisspeptin concentration; broken lines-% acetonitrile (ACN). ↓KP-52 represents the elution position of Kisspeptin-52 standard. The recovery of Kisspeptin-IR in the tissue extract from each column run was above 80% (means S.E.M.; n = 4).</p