The basal ganglia are a collection of sub-cortical nuclei involved in the execution of a range of motor and cognitive behaviours. The striatum is the input nucleus of the basal ganglia, receiving major excitatory innervation from the cerebral cortex and intralaminar thalamic nuclei. The main target of these two pathways are the principal striatal neurons, the medium-sized spiny neurons (MSNs), which are subdivided based on their axonal targets and the expression of molecular markers. Direct pathway neurons project to the output nuclei of the basal ganglia and express the D, dopamine receptor subtype, whereas indirect pathway MSNs project to the output nuclei via the globus pallidus, and express the D2 receptor. The striatum also contains interneurons that are essential in processing information within striatum; the cholinergic interneuron is of particular interest due to its role in reward-related behaviour. The aim of this study was to examine the cortical and thalamic innervation of subtypes of striatal neurons. To examine whether the cortical or thalamic afferents selectively innervate direct or indirect pathway neurons, transgenic mice expressing GFP under either the D, or D2 receptor promoter were used. Striatal sections from these mice were immunostained to reveal the GFP and selective markers of the cortical and thalamic afferents, VGluTI and VGluT2, respectively. A quantitative electron microscopic examination ofsynaptic connectivity was carried out. The results indicate that there is no selectivity of either the cortical or thalamic pathway for D, or D2 expressing MSNs. Thus both direct and indirect pathway MSNs are involved in the processing of both cortical and thalamic information The cortical and thalamic innervation to cholinergic interneurons was also examined. Stimulation of cortex and thalamus in vivo in anaesthetised rats resulted in short-latency excitatory responses in identified cholinergic interneurons, indicative of monosynaptic connections. After recording, cholinergic interneurons were filled with neurobiotin. The synaptic innervation from cortex and thalamus was then examined in two individual, electrophysiologically characterised, and neurochemically identified cholinergic interneurons. One neuron received input from both cortex and thalamus, whereas the other neuron received input from the thalamus only. These results provide anatomical and physiological data illustrating how the excitatory inputs to striatum innervate cholinergic interneurons.

Memoria para optar al Título Profesional de Médico Veterinario / El sinciciotrofoblasto presenta una membrana apical y una membrana basal, que constituyen la principal barrera para el intercambio materno fetal. El cloruro (Cl-) es el principal ión extracelular y su paso através de la membrana apical ha sido ampliamente estudiado. Sin embargo, en la membrana basal el transporte de cloruro ha sido poco estudiado. El presente trabajo es el primero en describir las vías conductivas de cloruro existentes en la membrana basal (MB) del sinciciotrofoblasto trasplantado a ovocitos de Xenopus leavis a través del método de Voltage clamp. Al transplantar MB a ovocitos de Xenopus Laevis, estos muestran un aumento de corrientes de al menos 3 veces en comparación a ovocitos controles y un aumento de conductancia de cuerda a 40 mV de 5,9 μS promedio. Además a 30 mV las corrientes exógenas disminuyeron en un 51,8% al agregar ácido 4,4'-diisotiocianatoestilbeno-2,2'-disulfónico (DIDS) y en un 52,8% al agregar ácido difenilamina-2-carboxílico (DPC), ambos conocidos bloqueadores de canales de Cl-. Estos datos demuestran la presencia vías conductivas para Cl- en la MB del sinciciotrofoblasto placentario humano / Financiamiento: Proyecto Fondecyt 1070695

Glutamic Acid Decarboxylase (GAD) is a rate-limiting enzyme responsible for synthesis of the inhibitory neurotransmitter GABA. Dopaminergic denervation in rodents by unilateral injections of 6-OHDA or MPTP causes an increase in Gad67 mRNA in the striatum, which is further exacerbated by administration of L-Dopa (Horvath et al., 2011; Katz et al., 2005 Bacci et al., 2002). Denervation of nigrostriatal neurons is the key pathological hallmark of Parkinson's disease, which results in hypokinetic movement and rigidity. Medium spiny projection neurons of the striatum comprise 95% of the neuronal population and utilize Gad67 (encoded by the Gad1 gene) for the synthesis of basal levels of GABA. The contribution of Gad67 to GABA signaling in medium spiny projection neurons in the striatum has not been thoroughly understood in normal or Parkinsonian states. Mice with a deletion in Gad67 in Gpr88 expressing neurons were generated by crossing mice with a floxed exon 2 of Gad1 with mice expressing Cre recombinase under the control of the Gpr88 promoter. The aim of this study was first, to characterize mice with a deletion in striatal Gad67 by immunohistochmical, electriophysiological and behavioral examination to determine whether Gad67 expression contributes to sensorimotor and learning tasks. And next, to investigate whether a downregulation in striatal Gad67 would decrease dyskinesia and affect the impaired motor symptoms following dopaminergic denervation with a unilateral 6-OHDA lesion and subsequent treatment with L-Dopa. In this study, neuronal Gpr88 expression was indicated by GFP reporter expression, which resulted from Cre-mediated excision of exon 2 of the Gad1 gene. Gpr88 expression was confirmed in the striatum, olfactory tubercle, cortex and brain stem. Furthermore, Gpr88 was confined to striatonigral and striatopallidal MSNs in the striatum. Additionally, Cre-mediated GFP reporter expression indicated that Gpr88 expression occurs throughout various brain regions, including the motor and visual areas of the cortex, amygdala, hippocampus and cerebellum during development. The developmental expression of Gpr88 seems to be a highly regulated process that occurs throughout the brain. In the conditional knockout mouse, deleting striatal Gad67 resulted in an upregualtion of Gad67 in the globus pallidus and downregulation in the substantia nigra. The changes in Gad67 expression indicate the effects of inactivating GABAergic signaling in striatonigral and striatopallidal MSNs in the direct and indirect pathways. Mice with a deletion in striatal Gad67 demonstrated compromised performance in spatial learning in the Morris water maze, suggesting that GABAergic striatal signaling in the direct and indirect pathways accounts for cue-based learning and spatial memory. However, inactivation of GABAergic signaling in striatonigral and striatopallidal MSNs does not account for motor deficits such as bradykinesia, akinesia or hypokinesia in intact mice; instead it perpetuates hyperkinetic motor activity. In the second experiment of this study, dopaminergic denervation by a unilateral 6-OHDA lesion induced bradykinesia and hypokinetic motor behavior, as demonstrated by impaired performance in the rota-rod and pole test. Additionally, L-Dopa administration to 6-OHDA lesioned mice evoked abnormal involuntary movements (AIMs) to the same degree in all dyskinetic mice. A deletion in striatal Gad67 did not decrease symptoms of dyskinesia, nor cause a lessening of motor impairment caused by dopaminergic denervation. Complete inactivation of the indirect pathway is believed to limit the inhibition of unwanted actions and may perpetuate dyskinesia, even when striatonigral MSNs of the direct pathway are inactive.

ABSTRACT Phenotypic flexibility in basal metabolic rate (BMR) in response to short-term thermal acclimation was assessed in the Laughing Dove (Streptopelia senegalensis), a common resident bird species distributed throughout most of southern Africa. I hypothesised that S. senegalensis would display flexibility in BMR over short time scales and that this flexibility would be reversible. Additionally, I hypothesised BMR to be repeatable, and that changes in BMR would be correlated with changes in organ mass. I tested these hypotheses by measuring BMR in three groups of 10 birds before and after a short-term (21 day) thermal acclimation period to one of three air temperatures (10o, 22o & 35oC). After acclimation the three temperature groups were randomly divided and reverseacclimated for another 21 days to one of the two thermal environments not yet experienced. After this reverse-acclimation period BMR was measured again. The dry masses of the stomach, kidney, heart, intestines, liver and pectoral muscles of acclimated birds were used to determine possible mechanistic correlates of BMR adjustments. Additionally, by monitoring BMR every 4-6 days during cold (10oC) and heat (35oC) acclimation I was able to assess the temporal dynamics of adjustments in BMR in response to short-term thermal acclimation. BMR was both flexible and reversible in S. senegalensis as a consistent relationship between BMR and acclimation air temperature was observed after acclimation and reverse-acclimation. BMR increased with decreasing acclimation temperature. Furthermore, a significant proportion (25%) of the observed variation in BMR was repeatable in the 22oC group in spite of the change in BMR induced by thermal acclimation. The mechanistic correlate of BMR adjustment in S. senegalensis appears to be metabolic intensity and not organ size, as the only organ to show a significant increase in size was the intestine of the acclimated 10oC group, which was significantly heavier than the intestine of the 22oC group. BMR also decreases in response to the reduction of flight and/or exercise. Since this reduction was not accompanied by a correlated change in organ mass or body mass, the reduction in BMR as a response to captivity appears to be linked to metabolic intensity of the organs and skeletal muscles. In S. senegalensis adjustments in BMR occur during the first 30 days of captivity and thermal acclimation. The response in BMR to acclimation temperature is clearly evident as BMR of the heat-acclimated group was significantly lower than the coldacclimated group after 21 days. During the response period, which lasts approximately 30 days, BMR adjusts as a mechanism to offset the costs of thermoregulation and habituation to captivity while other metabolic parameters such as body mass, body temperature, and minimum wet thermal conductance adjust to captivity and the thermal environment. After 30 days BMR of the cold and heat-acclimated groups converge on 0.68W, indicating that once the associated metabolic parameters adjust and stabilize in response to the thermal environment, BMR continues to adjust to captivity.

Introduction: Basal-like (BL) breast cancer is an aggressive sub-type of breast cancer for which there is no targeted systemic therapy. C-Met is a receptor tyrosine kinase implicated in breast cancer. Clinical trials assessing the efficacy of anti-c-Met therapy are underway, yet few studies have analysed the clinical significance of c-Met expression and/or activation in breast cancer, in particular whether there is a correlation with molecular sub-type. The aims of this study are: 1) to establish the clinical significance of c-Met expression in invasive breast cancer, 2) evaluate the novel proximity ligation assay (PLA) as a method of measuring c-Met activation and 3) address the effect of hepatocyte growth factor (HGF)-mediated c-Met phosphorylation on migration and protein expression in cell lines representative of the BL sub-type. Methods: Immunohistochemistry for c-Met was performed on 1455 cases of breast cancer using tissue microarray (TMA) technology. The PLA was performed on TMAs constructed from 181 breast cancers. C-Met expression and the PLA product were correlated with clinico-pathological parameters and survival. The effects of HGF on cell migration and protein expression were assessed using migration assays, western blots and immunofluorescent studies. Results: C-Met expression was independently associated with BL breast cancer (odds ratio = 6.44, 95% confidence interval (CI) = 1.74-23.78, p = 0.005) and reduced overall survival (hazard ratio = 1.81, 95% CI = 1.07-3.06), p = 0.026). The PLA signal was not associated with molecular sub-type or survival. HGF stimulation was associated with a significant increase in BL cell migration (p < 0.01) but no evidence of epithelial-mesenchymal transition was observed. Conclusion: My findings suggest BL breast cancer patients should be included in future trials of anti-c-Met therapy. Further work is necessary to establish the prognostic utility of the PLA as a measure of c-Met activation and the mechanisms driving HGF-mediated cell migration.

Subglacial processes that control the water pressure and flow determine the large-scale behavior of the overlying ice by regulating basal resistance. We implement a model in which a steady-state subglacial conduit system is surrounded by fully saturated porous media. We investigate branching in this system at fixed angles of 15 degrees, 30 degrees and 45 degrees to the direction of ice flow and further assess these systems by calculating the hydraulic potential gradient to determine conduit flow path. We solve our governing equations for porous media flow and allow ice infiltration of the pore space to occur at a critical effective stress N infiltration. For low values of N infiltration, ice infiltration of sediment allows these conduits to follow their original paths. Where insufficient ice infiltration occurs, the conduit path instead lies parallel to the direction of ice flow. Our results speak to the importance of incorporating small-scale processes into models of subglacial hydrologic networks.

Periocular malignancy represents an increasing burden and currently requires disfiguring surgery in an attempt to cure patients. Basal cell carcinoma (BCC) is the commonest cancer worldwide and morphoeic BCC (mBCC) is an aggressive subtype. Sebaceous gland carcinoma (SGC) is a rare, but life-threatening condition that often requires blinding surgery to prevent mortality, especially in the pagetoid subtype. MBCC has a high risk of local recurrence compared to the more indolent nodular subtype reflected by a different set of driver genes including FLNB and HECTD4. Surrounding mBCC stroma is abnormal, containing mutations in EPHA3 and GLI3. Four common dysregulated pathways detected using both whole exome and RNA sequencing for mBCC were; 'hedgehog (Hh) signalling pathway', 'BCC', 'Natural killer cell mediated cytotoxicity' and 'Fc Epsilon RI signalling pathway'. Hh mutational profile for nodular BCC was not reflected in the RNA and protein expression. In contrast, Hh overexpression is seen in the tumour and stroma of morphoeic tissue with the latter potentially being partly responsible for its aggressive nature and risk of recurrence that may warrant removal to prevent recurrence. SGC has a low overall mutational burden, no UV signature and defective mismatch repair signature. Driver genes included TP53, RB1 and the dynein family is a novel driver possibly involved in chromatid segregation as marked chromosomal instability was demonstrated on copy number analysis. Correlation of whole exome and RNA sequencing data demonstrated upregulated 'cell cycle', 'ubiquitin mediated proteolysis' and 'wnt signalling'. Subtype analysis of pagetoid and nodular SGC revealed the histone gene cluster family as important to both. Oncomir hsa-miR-21 was overexpressed in both and loss of hsa-miR-199a occurs in pagetoid. Increased protein expression of HIST1H2BD was seen in both subtypes as was Hh expression. These novel SGC findings support a chromosomally unstable cancer with the ability to invade extracellular matrix.

Breast cancer is the most common cancer in the UK and Basal-like breast cancer (a highly aggressive subtype) accounts for approximately 8-22% of all cases depending on ethnicity. Unlike most human malignancies and indeed other PAM50 breast cancer subtypes, the vast majority of Basal-like tumours are positive for wild type p16. This p16 signature is associated with a particularly poor prognosis and p16-positive Basal-like breast cancer remains the most clinically challenging subtype and is the focus of this project. Pro-senescence therapies are gaining momentum as attractive strategies for the treatment of those breast cancers with current unmet clinical need. To identify targets for pro-senescence therapy in p16-positive Basal-like breast cancer, a genome‐wide siRNA screen and two subsequent validation screens using two p16-positive cancer cell lines were performed. Screening revealed 20 siRNAs that induced senescence within both cancer cell lines. Strikingly, 11 of these 20 siRNAs targeted ribosomal proteins, implicating disrupted ribosomal biosynthesis in senescence activation in p16-positive Basal-like breast cancer. Importantly, subsequent experiments in normal human mammary epithelial cells established that specific ribosomal protein knockdown is well tolerated by normal cells. Analysis of the METABRIC data set showed a high degree of ribosomal dysregulation in Basal-like tumours and revealed that all 11 ribosomal hits identified were frequently overexpressed in p16-positive Basal-like breast cancers. Kaplan Meier analysis confirmed that elevated expression of six of the 11 ribosomal proteins correlates with a reduced overall survival in these women, further supporting a role for these proteins as drivers of disease. These six ribosomal hits, associated with the poorest patient survival, were prioritised for further validation. Senescence induction was found to be highly stable, and associated with dramatic changes to nucleolar morphology, reminiscent of the nucleolar signature observed upon premature senescence induction in normal human mammary epithelial cells. In addition, siRNA rescue experiments indicated that senescence initiation is dependent on p16 and p21 expression and is accompanied by p16 nuclear translocation and p21 degradation. Further, ribosomal protein silencing in MDA-MB-231 cells (p16-null Basal-like breast cancer cell line) resulted in a 'death-like' phenotype, partially dependent on p21 expression suggesting that, within a cancer context, ribosomal protein silencing may induce a differential response depending on the status of p16. In conclusion, it is proposed that these six ribosomal candidates may form the basis of a novel pro-senescence therapy for p16-positive Basal-like breast cancer. They may also represent novel prognostic biomarkers for this disease subset and may help to improve disease stratification and future directed personalised therapies.

Att använda inkontinensskydd innebär ett lidande för den äldre patienten och en betydande kostnad för samhället. Detta kan med olika insatser i omvårdnad påverkas och förbättras. Urininkontinens är ett problem där prevalensen tenderar öka då äldre personer flyttar in på vård- och omsorgsboende. Efter inflyttning till vård- och omsorgsboende ska patienten få en vård- och genomförandeplan upprättad, gällande insatser som patienten är i behov av. Syftet med studien är att undersöka hur patientens problematik med urininkontinens dokumenteras i vårdplanen och hanteras på vård – och omsorgsboende i genomförandeplanen. Frågeställningar som är kopplade till syftet: Hur journalförs och dokumenteras patientens urininkontinens i vårdplanen? Hur är omsorgsbehovet i genomförandeplanen utformat? Och hur relateras vårdplanen till genomförandeplanen? Metoden som användes i studien är en kvalitativ innehållsanalys med induktiv ansats av vård - och genomförandeplaner. Vidare undersöktes genom granskning av dessa dokument om överensstämmelse förekommer mellan dem. Resultatet visar att flertalet patienter inte har en upprättad vård- och genomförandeplan om urininkontinens och hur omvårdnaden kring patientens besvär med urininkontinens ska utformas. Samstämmigheten mellan vård- och genomförandeplaner visar på att det finns brister. Vidare visas behovet av basala utredningar och tydligare beskrivningar av omvårdnadsåtgärder. Det finns ett behov av att utveckla omvårdnaden av patienter med urininkontinens genom utbildning och utveckling av arbetsrutiner. Tvärprofessionellt samarbete och arbete med olika kvalitetsregister kan utvecklas och förbättras

Basal-like breast cancer represents a subgroup of mammary cancers associated with a particularly poor prognosis, as they are refractory to current targeted therapies employed for the treatment of breast cancer. In this work I aimed to explore the therapeutic potential of selective estrogen receptor modulators (SERMs), a targeted breast cancer treatment, in combination with epigallocatechin gallate (EGCG), for the treatment of basal-like breast cancer, using MDA-MB-231 cell as an in vitro model of the disease. A significant reduction in MDA-MB-231 cell number and a significant increase in cytotoxicity was observed following treatment with 25 [mu]M of EGCG in combination with 1 [mu]M of 4-hydroxytamoxifen (4-OHT) (EGCG+4-OHT) or 4 [mu]M of raloxifene (EGCG+Ral) over a 36 h time course. However, these effects were not resolved in time, with an increase in G₁-phase cell cycle arrest. Changes in the metabolism of EGCG were dismissed as a possible mechanism through which the combination treatments may be eliciting the cytotoxicity. Changes in the expression and phosphorylation of various signaling proteins, important for the proliferation and survival of basal-like breast cancer, were investigated through Western blotting. Interestingly, the two combination treatments produced very similar results; reductions in the phosphorylation of EGFR and AKT occurred after 6, 12, and 18 h with EGCG+4-OHT and 6, 12, 18 and 24 h with EGCG+Ral, while a reduction in S6K phosphorylation was observed following 6, 12, 18 and 24 h of both combination treatments. Interestingly, both SERMs contributed significantly to the net reduction in S6K phosphorylation, induced by the combination treatments. Both combination treatments were also associated with a significant increase in the phosphorylation and total expression of stress activated protein kinases, p38 and JNK1/2 following 12, 18 and 24 h of treatment. As changes were observed at an intracellular signaling level, the effect of the combination treatments were investigated at the transcriptomic level after 18 h of treatment, using human oligonucleotide microarrays. This transcriptomic analysis revealed that both combination treatments reduced the transcript expression of five enzymes involved with cholesterol synthesis, which was confirmed through qRT-PCR. Cholesterol is an important component of the plasma membrane and is critical for the transduction of extracellular signals. Furthermore, both combination treatments induced the transcriptomic expression of the zinc coordinating metallothionein (MT) proteins. This was associated with an increased nuclear localization of MTF-1, the transcription factor responsible for MT expression, after 6, 12 and 18 h of both combination treatments. Finally, nuclear Western blotting of the NF-[kappa]B subunit, p65, revealed that both combination treatments reduced the nuclear localization of NF-[kappa]B following 6, 12 and 18 h. In collating this data, it appears that the combination treatments of EGCG+4-OHT and EGCG+Ral are inducing cytotoxicity through various mechanisms, including reduced cellular signaling through EGFR, AKT and S6K, increased stress signaling through JNK1/2 and p38 and altered gene expression of MTs and enzymes involved with cholesterol synthesis. Therefore, the combination treatment of EGCG+SERMs exhibits therapeutic potential in MDA-MB-231 cells, a model of basal-like breast cancer.