Yes / Medical Research Council, Innovate UK and Epistem Ltd.

Kidney disease is a major complication of diabetes and accounts for one-third of all diabetes-related deaths. Estrogen is considered protective against cardiovascular and non-diabetic renal disease, however it is unclear if this protection extends to diabetes and diabetic kidney disease.To address these questions, we have used a new model of menopause in which repeated daily injections of 4-vinylcyclohexene (VCD) induces gradual ovarian failure in mice. Unlike with ovariectomy, the VCD model preserves the gradual transition into ovarian failure (OF) (modeling perimenopause). Also, following OF, the residual ovarian tissue is retained and secretes androgens, similar to the androgen production by postmenopausal human ovaries.The VCD model of menopause was combined with the streptozotocin (STZ) model of type 1 diabetes, and the development of diabetes and diabetic kidney damage were studied over the subsequent 6 weeks. We observed that blood glucose levels are higher in post-OF diabetic mice compared to cycling diabetic and peri-OF diabetic mice. Renal cell proliferation, an early marker of kidney damage, is increased in post-OF diabetic mice compared to cycling diabetic mice, as measured by expression of proliferating cell nuclear antigen. We also demonstrate that expression of α-smooth muscle actin is increased in post-OF diabetic mice compared to cycling diabetic mice. Five weeks after STZ injection, post-OF diabetic mice had higher rates of urine albumin excretion than cycling diabetic mice.Using real-time PCR, we identified changes in expression between post-OF diabetic and cycling diabetic mice of genes which have previously been associated with diabetic kidney damage. We also show that some of these changes occur in peri-OF diabetic mice as well. Using microarray, we identified 119 new genes which are regulated by the combination of ovarian failure and diabetes in the mouse kidney.These data support our hypothesis that the changes in hormones which occur during the transition into ovarian failure exacerbate the development and progression of diabetic kidney damage in mice. These data also highlight the utility and importance of the VCD model of menopause in the study of diabetic kidney damage.

Metallic glass matrix composites have enormous potential stemming from the interplay between crystalline and amorphous phases. This work models such a composite using shear transformation zone dynamics (a modified kinetic Monte Carlo method) for the amorphous phase, and a local Taylor dislocation model for the crystalline phase. An N-factorial experiment using the model is presented examining the effects of crystalline volume fraction, microstructure length scale, and yield stress of the crystalline phase. Each replicate is analyzed for maximum stress, maximum strain, strain energy dissipation, strain localization, and strain partitioning between phases. Regression analysis is used to identify statistically-significant trends in the data. The experiment shows that strain delocalization and the consequent ductility are facilitated by a crystalline phase with a substantially lower yield stress than that of the amorphous matrix. It also shows that increasing crystalline volume fraction alone is insufficient to promote strain delocalization in the case of a crystalline phase with high relative yield stress, and that a lower yield stress for the crystalline phase implies lower maximum stresses supported by the composite. Therefore designers must balance the need for ductility and delocalization against the composite yield stress by finding an optimal combination of volume fraction and crystalline mechanical properties. This work provides continuous functional forms for the relationships between these properties to aid in that optimization process.

Orientador: Débora Cristina Damasceno / Banca: Marilza Vieira Cunha Rudge / Banca: Estela Maris Andrade Forell Bevillacqua / Banca: Renée Laufer Amorim / Banca: Teresa Cristina França Sartori / Resumo: Avaliar os efeitos do diabete moderado nos parâmetros reprodutivos maternos e no desenvolvimento placentário-fetal em ratas Wistar. Metodologia: No dia do nascimento, 147 ratas Wistar foram distribuídas aleatoriamente em dois grupos experimentais: Grupo não-diabético (Controle, n=45) - recebeu o veículo; Grupo diabético (STZ, n=102) - recebeu 100 mg streptozotocin/kg. Na fase adulta, as ratas foram acasaladas e, no dia 0 de prenhez, foram incluídas ratas controle que apresentassem glicemia abaixo de 120 mg/dL e, para o grupo diabete moderado, glicemia entre 120 e 300 mg/dL. Em diferentes momentos da prenhez, glicemia e peso corpóreo foram verificados. No 21º dia de prenhez, as ratas foram anestesiadas para coleta de sangue para dosagem de insulina e, em seguida, foi realizada laparotomia para retirada e pesagem dos fetos e placentas. Os dados maternos e fetais foram analisados por Twoway ANOVA seguida do Teste t. Os recém-nascidos (RN) foram classificados em pequenos, adequados e grandes para idade de prenhez e as comparações entre os grupos foram realizadas segundo o Teste de Qui-quadrado. As ratas STZ apresentaram glicemias maiores nos dias 0 e 14 de prenhez, menor número médio de fetos vivos, implantações e de corpos lúteos, aumento nas taxas de perdas embrionárias pós-implantação, no peso placentário e na proporção de RN pequenos (PIP) e grandes (GIP) para idade de prenhez, redução de RN AIP e inalteração nas concentrações de insulina. Portanto, o diabete de intensidade moderada alterou a glicemia materna no início da prenhez, que deflagrou alterações no organismo materno e/ou no desenvolvimento inicial do embrião, afetando sua implantação e futuro desenvolvimento placentário e fetal. / Abstract: To evaluate the mild diabetes effects on the maternal reproductive outcome and placental-fetal development in female Wistar rats. Methodology: At the birth day, 147 female rats were randomly distributed in two experimental groups: 1) Non-diabetic Group (Control, n=45) - received the vehicle; 2) Diabetic Group (STZ, n=102) - received 100 mg streptozotocin/kg. At the adult phase, the female rats were mated and, at the day 0 of pregnancy, they were included in the control group when presented glycemia below 120 mg/dL and, in the group STZ when showed glycemia between 120 and 300 mg/dL. In different moments of the pregnancy, glycemia and body weight were verified. At day 21 of pregnancy, the rats were anaesthetized to collect blood samples for insulin determination and, soon afterwards, the laparotomy was carried out to withdraw and weigh the fetuses and placentas. The maternal and fetal dates were analyzed by Two-way ANOVA followed by t Test. The newborns (NB) were classified in small, appropriate and large for gestational age and the comparisons between the groups were accomplished according to Qui-square Test. Rats STZ presented higher glycemia at days 0 and 14 of pregnancy, lower numbers of alive fetuses; implantations and corpora lutea; increased rate of embryonic losses, placental weight and proportion of small NB (SGA) and large (LGA); reduced rate of AGA NB and unaltered insulin concentrations. Therefore, the mild diabetes altered the maternal glycemia in the early pregnancy, which caused changes in the maternal organism and/or in the early development of the embryo, impairing its implantation and future placental and fetal development. / Doutor

The phenomenon of vibration-induced shear resistance reduction (ViSRR) in granular soil is characterized by the loss of shear resistance without significant excess pore pressure generation. It has diverse potential applications in various industries such as mining, pharmaceuticals, and civil engineering, including the installation of vibratory-driven piles. Despite limited research on this topic, both experimentally and theoretically, the mechanism associated with ViSRR remains challenging to explain. There is currently no established constitutive model to properly describe it. This dissertation investigates the fundamental features of ViSRR and develops a model to describe the process that leads ViSRR. To achieve these objectives, three main areas of investigation were undertaken. First, a series of laboratory tests were conducted using a modified triaxial apparatus that allowed for vibrations superimposed on the monotonic shearing of granular soil samples. Second, by correlating macroscopic plastic strains with the transition, creation, and destruction of mesoscopic shear-transformation-zones (STZs), which can be considered as weak particle loops in granular assemblies, the conventional thermodynamic-based STZ model was extended to soil mechanics. Third, the concept of "vibration-induced shear resistance relaxation" was proposed, which refers to the loss of shear resistance in granular material subjected to restricted deformations in response to plastic strains induced by vibrations. In other words, ViSRR occurs when the total deformation rate of the granular material is constrained and does not keep up with the rate of plastic deformation induced by vibrations. By conducting laboratory tests, developing the extended STZ model, and proposing the concept of "vibration-induced shear resistance relaxation", this dissertation contributes to a better understanding of ViSRR in granular soil and provides insights into the mechanisms governing this phenomenon. The results of this research can be used to improve the design and construction of geotechnical structures. / Thesis / Doctor of Philosophy (PhD)

La rétinopathie diabétique est une cause majeure de cécité et de malvoyance qui affecte jusqu'à 90% des patients atteints de diabète. Le Maroc n’échappe pas à cette pathologie, qui est connue pour altérer le fonctionnement du système visuel et pourrait conduire également à des désordres chronobiologiques, aussi bien chez l’Homme que chez des modèles animaux. Ces altérations pourraient être liées aux dégénérescences neuronales des systèmes de photoréception classique (cône et bâtonnet) et des cellules ganglionnaires à mélanopsine, impliqués dans la régulation et l’entraînement par la lumière du système circadien. Cependant, à l’heure actuelle, peu d’études ont analysé précisément l’impact de la rétinopathie diabétique sur le système circadien. L’objectif de notre travail est d’analyser au cours de la rétinopathie diabétique (1) l’atteinte des cônes, des bâtonnets et des cellules ganglionnaires à mélanopsine, (2) le fonctionnement endogène moléculaire et la réponse à la lumière des horloges centrale et rétinienne et (3) la réponse comportementale du système circadien à la lumière. Notre stratégie est basée sur l’utilisation d’un modèle murin, chez lequel le diabète est induit expérimentalement par l’administration d’un agent chimique la streptozotocine (STZ), toxique pour les cellules β pancréatiques. Des approches morphométriques, moléculaires et comportementales ont été utilisées. Nos résultats montrent que le diabète induit des changements morphologiques des cellules ganglionnaires à mélanopsine tels que des gonflements des somas et des varicosités au niveau des dendrites avec une préservation du nombre total de ces cellules. Ceci est associé à une diminution de l’induction par la lumière du gène c-fos et des gènes de l’horloge Per1 et Per2 au niveau du SCN et à l’absence de cette induction au niveau rétinien au stade 12 semaines après l’induction du diabète. La machinerie moléculaire des horloges rétinienne et centrale évaluée par l’analyse de l’expression circadienne des gènes de l’horloge et des gènes contrôlés par les gènes de l’horloge montre que certains gènes de l’horloge clés pour chaque tissu sont altérés. A l’échelle comportementale, les souris STZ (souris diabétiques) montrent une réduction de l’amplitude du rythme de leur activité locomotrice totale et une diminution de la sensibilité à la lumière aux faibles intensités. Après une avance de phase du cycle 12L/12D, ces animaux présentent également une diminution de la vitesse de resynchronisation au nouveau cycle lumineux imposé par rapport aux animaux témoins. Ces nouvelles données montrent que le diabète de type 1 altère les réponses du système circadien à la lumière d’un point de vue moléculaire et comportemental et suggèrent que les patients diabétiques peuvent présenter des troubles circadiens particulièrement lorsqu’ils sont soumis aux challenges chronobiologiques / Diabetic retinopathy is a major cause of blindness and is commonly viewed as a vascular complication of type 1 diabetes. However, this kind of diabetes causes visual dysfunction before the onset of clinically visible microvascular changes, associated with diabetic retinopathy. Several histopathological studies in diabetic patients and in chemically-induced or genetic rodent models of diabetes indicate that photoreceptors and retinal ganglion cells (RGCs) are affected by diabetes with apoptotic degeneration. There is increasing evidence that melanopsin-expressing ganglion cells that are crucial for the regulation of a range of non-visual functions including the photic synchronization of circadian rhythms are altered in retinal pathologies. The link between diabetes and circadian rhythms has only been addressed in a relatively limited number of studies. Using a streptozotocin-induced (STZ) model of diabetes, we investigated the impact of diabetic retinopathy on non-visual functions by analyzing the morphology of melanopsin ganglion cells and light-induced c-fos and Period 1-2 clock genes in the central (SCN) and the retina clocks. The effect of this pathology on the endogenous circadian function of clock and controlled clock genes was assessed in the SCN and the retina at 12 weeks post-diabetes. Behaviorally, the ability of STZdiabetic mice to entrain to light was challenged by the exposure of animals to 1) successive light/dark (LD) cycle of decreasing or increasing light intensities during the light phase and 2) 6-hr advance of the LD cycle. Our results show that diabetes induces morphological changes of melanopsin-expressing ganglion cells including soma swelling and dendritic varicosities with no reduction in their total number, associated with decreased c-fos and clock genes induction by light in the SCN and also in the retina at 12 weeks post-onset of diabetes. In addition, the circadian expression of major clock genes was altered in the central and retinal clocks, suggesting that RD affects the endogenous molecular machinery and the light response of these two clocks. Moreover, STZ-diabetic mice exhibited a reduction of overall locomotor activity, a decrease of circadian sensitivity to light at low intensities, and a delay in the time to re-entrain after a phase advance of the LD cycle. These novel findings demonstrate that diabetes alters clock genes and behavioral responses of the circadian timing system to light and suggest that diabetic patients may show an increased propensity for circadian disturbances, in particular when they are exposed to chronobiological challenges

Made available in DSpace on 2014-06-11T19:35:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-27Bitstream added on 2014-06-13T18:47:00Z : No. of bitstreams: 1 sinzato_yk_dr_botfm.pdf: 273220 bytes, checksum: 7f61ff6bd8aff6dcd2d1543cbfa1ca95 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Avaliar os efeitos do diabete moderado nos parâmetros reprodutivos maternos e no desenvolvimento placentário-fetal em ratas Wistar. Metodologia: No dia do nascimento, 147 ratas Wistar foram distribuídas aleatoriamente em dois grupos experimentais: Grupo não-diabético (Controle, n=45) - recebeu o veículo; Grupo diabético (STZ, n=102) - recebeu 100 mg streptozotocin/kg. Na fase adulta, as ratas foram acasaladas e, no dia 0 de prenhez, foram incluídas ratas controle que apresentassem glicemia abaixo de 120 mg/dL e, para o grupo diabete moderado, glicemia entre 120 e 300 mg/dL. Em diferentes momentos da prenhez, glicemia e peso corpóreo foram verificados. No 21º dia de prenhez, as ratas foram anestesiadas para coleta de sangue para dosagem de insulina e, em seguida, foi realizada laparotomia para retirada e pesagem dos fetos e placentas. Os dados maternos e fetais foram analisados por Twoway ANOVA seguida do Teste t. Os recém-nascidos (RN) foram classificados em pequenos, adequados e grandes para idade de prenhez e as comparações entre os grupos foram realizadas segundo o Teste de Qui-quadrado. As ratas STZ apresentaram glicemias maiores nos dias 0 e 14 de prenhez, menor número médio de fetos vivos, implantações e de corpos lúteos, aumento nas taxas de perdas embrionárias pós-implantação, no peso placentário e na proporção de RN pequenos (PIP) e grandes (GIP) para idade de prenhez, redução de RN AIP e inalteração nas concentrações de insulina. Portanto, o diabete de intensidade moderada alterou a glicemia materna no início da prenhez, que deflagrou alterações no organismo materno e/ou no desenvolvimento inicial do embrião, afetando sua implantação e futuro desenvolvimento placentário e fetal. / To evaluate the mild diabetes effects on the maternal reproductive outcome and placental-fetal development in female Wistar rats. Methodology: At the birth day, 147 female rats were randomly distributed in two experimental groups: 1) Non-diabetic Group (Control, n=45) - received the vehicle; 2) Diabetic Group (STZ, n=102) - received 100 mg streptozotocin/kg. At the adult phase, the female rats were mated and, at the day 0 of pregnancy, they were included in the control group when presented glycemia below 120 mg/dL and, in the group STZ when showed glycemia between 120 and 300 mg/dL. In different moments of the pregnancy, glycemia and body weight were verified. At day 21 of pregnancy, the rats were anaesthetized to collect blood samples for insulin determination and, soon afterwards, the laparotomy was carried out to withdraw and weigh the fetuses and placentas. The maternal and fetal dates were analyzed by Two-way ANOVA followed by t Test. The newborns (NB) were classified in small, appropriate and large for gestational age and the comparisons between the groups were accomplished according to Qui-square Test. Rats STZ presented higher glycemia at days 0 and 14 of pregnancy, lower numbers of alive fetuses; implantations and corpora lutea; increased rate of embryonic losses, placental weight and proportion of small NB (SGA) and large (LGA); reduced rate of AGA NB and unaltered insulin concentrations. Therefore, the mild diabetes altered the maternal glycemia in the early pregnancy, which caused changes in the maternal organism and/or in the early development of the embryo, impairing its implantation and future placental and fetal development.

No description available.

Metallic glass matrix composites (MGMCs) have been developed to improve upon the ductility of monolithic metallic glass. These composites utilize a secondary crystalline phase that is grown into an amorphous matrix as isolated dendritic trees. This work seeks to understand the mechanisms underlying strain delocalization in MGMCs in order to better direct efforts for continual progress in this class of material. A mesoscale modelling technique based on shear transformation zone (STZ) dynamics is used to do so. STZ dynamics is a coarse grained technique that can provide insight into the microscopic processes that control macroscopic behavior, but which can be difficult to resolve experimentally. A combined simulated-experimental approach to extract the individual material properties of the amorphous and crystalline phases is presented. Numerically, STZ dynamics is used to simulate nanoindentation of the crystalline and amorphous phases respectively. The indented phases are modelled as discs with varying thickness embedded in the other phase. Indentation depths are held constant. Experimentally, nanoindentation is carried out on DH2 and DH3 MGMC composites under varying loads at Stony Brook University (SBU). Specimens are cross-sectioned and using scanning electron microscopy, indentation sites are chosen so that the indenter targets individual phases. For both experimental and simulated nanoindentation, hardness and modulus values are calculated from the load-displacement data. The experimental and simulated values are normalized and compared. Good agreement between results suggests accurate characterization of the individual phases at low loads on both DH2 and DH3 composites. Length scales at which indentations begin sampling outside the intended phase are presented. Work is then presented on simulated uniaxial tensile loading of MGMCs. Dendritic microstructural sizes are varied and shear banding characteristics are measured. A competition of shear band nucleation and propagation rates that previously had only been seen in monolithic metallic glasses under certain loading conditions is found to exist in MGMCs as well. The stages of shear banding in MGMCs are presented and the influence of dendrites on shear band nucleation and propagation are discussed. It is proposed that the introduction of dendrites into the amorphous matrix work to inhibit shear band propagation and encourage shear band nucleation to delocalize strain in MGMCs. In particular, it was found that smaller dendrite sizes and spacings are better at doing so.

My PhD research studies, described in this thesis, were designed to document the temporal development of mechanical allodynia, a hallmark symptom of painful diabetic neuropathy (PDN), as well as opioid hyposensitivity using two different rat models of diabetes mellitus (DM). Specifically, the studies were conducted using the streptozotocin (STZ)-diabetic rat model of chemically-induced Type 1 diabetes in two different rat strains, as well as the Zucker Diabetic Fatty (ZDF) rat genetic model of Type 2 diabetes. Additionally, a longitudinal investigation of the effect of basal insulin replacement therapy to restore euglycaemia from 7-days post-STZ administration, on the development of mechanical allodynia in the hindpaws of the STZ-diabetic Wistar rat model of PDN, was conducted. The studies herein also included a longitudinal study to document the temporal development of mechanical allodynia and opioid hyposensitivity in the ZDF rat, which also examined the influence of dietary composition on the time course for the development of mechanical allodynia in the hindpaws, together with opioid hyposensitivity in these animals. In the final section of this thesis, the experiments were designed to examine possible mechanisms that may contribute to the development of opioid hyposensitivity in ZDF diabetic rats. These experiments involved the quantification of opioid receptor messenger ribonucleic acid (mRNA) gene expression as well as μ-opioid receptor (MOP-r) functional responses in tissues collected from 29-wk old diabetic ZDF rats relative to 7-wk old pre-diabetic control ZDF animals. In Chapter One, diabetes mellitus and more specifically its longterm complication, PDN, the focus of this doctoral research program, has been reviewed. Specifically, possible pathogenic mechanisms underlying mechanical allodynia, the relevant diabetic rodent models of PDN, use of insulin replacement therapy in diabetic rodents and its impact on hallmark symptoms of PDN, role of opioid pharmacology, the comparative efficacy of opioids in the treatment of PDN, and possible mechanisms that may underpin the development of opioid hyposensitivity in PDN, including the impact of altered excitatory neurotransmitters, have been reviewed. In Chapter Two, a preliminary study was conducted to investigate the efficacy of 4-wks treatment with Linplants (subcutaneous (s.c.) sustained-release bovine insulin implants) alone and in combination with ActRapid® (s.c. human insulin; 0.05 U to 3.5 U/100 g/day) with respect to glycaemic control in STZ-diabetic Wistar rats, and on acute diabetes characteristics for a 5-wk post-STZ administration period. Briefly, STZ-diabetic rats were divided into three groups: (1) rats which received no insulin treatment, (2) rats which were implanted with one s.c. Linplant at Day 7 post-STZ administration, and (3) rats which received one s.c. Linplant plus a once-daily injection of ActRapid® once diabetes was confirmed at 7-days post-STZ administration. The findings were that following implantation of a single Linplant at Day 7 post-STZ administration, euglycaemia was achieved in 50% of STZ-diabetic rats, with glycaemic control maintained for up to 4-wks post-implantation. Furthermore, once-daily injection of ActRapid™ to animals whose blood glucose levels (BGLs) were not well-controlled through use of Linplants alone, failed to achieve euglycaemia. It is possible that the ActRapid™ doses administered were not sufficient to achieve euglycaemia, and that increasing the doses may provide more effective glycaemic control. However, doubling the mean ActRapid™ dose from 1.63 (+ 0.3) U administered at Day 28 to 2.56 (+ 0.6) U administered at Day 34 post-STZ administration effectively only reduced BGLs by 1.3 mM to 11.6 + 1.6 mM. This suggests that although administering additional large doses of ActRapid™ to STZ-diabetic rats may eventually achieve euglycaemia, this method would presumably not be a more efficient method in achieving euglycaemia compared with the use of dosage-adjustable s.c. Linplants. Group (1) STZ-diabetic rats which were not treated with insulin developed diabetic signs including polydipsia, hyperphagia, decreased rate of body weight gain, and mechanical allodynia. Group (2) rats in which insulin treatment from 7-days post-STZ administration restored euglycaemia and reversed polydipsia and hyperphagia, were protected against the development of mechanical allodynia and reduced weight gain for the 5-wk study duration, while rats from Group (3) with incomplete glycaemic control developed levels of polydipsia, hyperphagia, reduced weight gain and mechanical allodynia intermediate between rats in Groups (1) and (2). These findings collectively suggest a direct correlation between the level of glycaemic control and the extent to which mechanical allodynia, a defining symptom of PDN, develops. In Chapter Three, the findings from the preliminary 5-wk study in Chapter Two were used to design a 24-wk longitudinal study of the temporal development of mechanical allodynia and opioid hyposensitivity in STZ-diabetic Wistar rats for comparison with the findings of a similar study previously undertaken by our laboratory using STZ-diabetic Dark Agouti rats (Nielsen et al, 2007). Additionally, this study examined the effects of tight glycaemic control achieved through the use of insulin implants as a means of potentially preventing the development of mechanical allodynia and opioid hyposensitivity for up to 24 weeks in STZ-diabetic Wistar rats. Briefly, STZ-diabetic rats were divided into 3 groups: (1) non-insulin treated STZ-diabetic Dark Agouti rats to provide comparison data with our laboratory’s previously published data in this rat strain (Nielsen et al, 2007), (2) non-insulin treated STZ-diabetic Wistar rats to examine possible between-species differences, and (3) STZ-diabetic Wistar rats which were treated with adjustable-dose s.c. Linplants from Day 7 post-STZ administration to maintain euglycaemia for the remainder of the 24-wk study period. In this 24-wk longitudinal study in STZ-diabetic rats, body weight, 24-hr water intake, paw withdrawal thresholds (PWTs) and BGLs were monitored at fortnightly intervals in all animals in order to document possible temporal changes in the development of diabetic signs and mechanical allodynia in the hindpaws respectively. STZ-diabetic rats underwent 6-wkly opioid antinociceptive testing, using single bolus doses of each of morphine and oxycodone with a 2-3 day washout period between individual opioids in order to assess the potential influence of both diabetes and glycaemic control on opioid potency in these animals. The findings demonstrate that non-insulin treated STZ-diabetic rats of both strains exhibited a decreased rate of body weight gain and polydipsia, as well as progressive development of mechanical allodynia in the hindpaws and loss of morphine potency. Importantly, STZ-diabetic Wistar rats which were treated with insulin to maintain euglycaemia from Day 7 post-STZ administration failed to develop these diabetic symptoms for the duration of the 24-wk study period, highlighting the importance of chronic hyperglycaemia in the development of mechanical allodynia and morphine hyposensitivity in the STZ-diabetic rodent model of PDN. The research described in Chapter Four involved a 22-wk longitudinal study of the development of diabetes and its longterm sensory nerve complications, viz mechanical allodynia and opioid hyposensitivity, in the ZDF rodent model of Type 2 diabetes commencing at 7-wks of age. This study also examined the influence of four different diets fed to separate groups of ZDF rats from 7-wks age, on the time course for the development of diabetes, mechanical allodynia in the hindpaws and opioid hyposensitivity in these animals. Briefly, ZDF rats were sub-divided into four dietary groups, each of which was fed one of the four following diets for 22-wks commencing at 7-wks of age, viz: (a) Purina 5008™, (b) a domestically-produced rat chow of similar composition to Purina 5008 (termed Purina Composition diet), (c) a Diabetogenic diet, or (d) Standard Rat Chow. All rats underwent once-fortnightly measurement of BGLs, body weight, 24-hr water intake, and measurement of PWTs in the hindpaws. Additionally, ZDF rats underwent opioid antinociceptive testing, similar to that previously described for STZ-diabetic rats (Chapter Three), to investigate the influence of diabetes and dietary composition on the antinociceptive potency of single bolus doses of morphine and oxycodone administered at 6-weekly intervals over a 22-wk study period. The afore-mentioned data were compared with the respective data obtained from the pre-diabetic control group of ZDF rats that were euthanised at 7-wks of age prior to the development of hyperglycaemia. The results demonstrate that the ZDF rat develops mechanical allodynia in the hindpaws and opioid hyposensitivity in a temporal fashion, in a manner similar to that previously documented for the STZ-diabetic Wistar rat model of Type 1 diabetes (Chapter Three). For the four diets assessed, there did not appear to be significant differences between dietary groups with respect to the time course and extent of development of hyperglycaemia, mechanical allodynia or opioid hyposensitivity in the ZDF rat model of PDN. The study described in Chapter Five investigated the effect of both diabetes and dietary composition on opioid receptor mRNA expression in tissue samples collected from the five groups of ZDF rats used in the behavioural studies described in Chapter Four and outlined above. Briefly, mRNA expression for each of the - (MOP), - (DOP), and - (KOP) receptors were quantified in mid-brain and spinal cord tissues prepared from 29-wk old diabetic ZDF rats maintained on one of four diets from 7-wks age, and compared with the respective expression levels in samples prepared from pre-diabetic ZDF rats euthanised at 7-wks of age. Overall, the findings suggest that diabetes does not alter opioid receptor mRNA expression in the mid-brain or spinal cord of diabetic ZDF rats at 29-wks of age relative to the corresponding levels of mRNA expression in the mid-brain and spinal cord of pre-diabetic ZDF rats at 7-wks of age. Hence, the marked reduction in the anti-allodynic potency of morphine and oxycodone observed in diabetic ZDF rats at 29-wks of age relative to that observed in pre-diabetic ZDF rats at 7-wks of age (Chapter Four) does not appear to be associated with a decrease in opioid receptor mRNA expression. In Chapter Six, the effect of both advanced diabetes and dietary composition on opioid-agonist stimulated [35S]GTPγS binding was examined in spinal cord tissue membranes from the ZDF rat. Specifically, [35S]GTPγS binding assays were used to assess the ability of a -opioid ligand (DAMGO) to stimulate -opioid receptor coupling to inhibitory G proteins in homogenates prepared from spinal cord samples of 29-wk old ZDF rats maintained on one of four different diets from 7-wks age (Chapter Four), relative to [35S]GTPγS binding in homogenates prepared from spinal cord samples of pre-diabetic 7-wk old ZDF rats. As specific MOP agonist-stimulated [35S]GTPγS binding was significantly decreased in spinal cord homogenates from diabetic ZDF rats at 29-wks of age relative to that for pre-diabetic ZDF rats (7-wks), this may contribute, at least in part, to the morphine hyposensitivity observed in diabetic ZDF rats at 29-wks of age relative to the pre-diabetic ZDF group. However, closer examination of these data revealed that specific MOP agonist-stimulated [35S]GTPγS binding above basal did not differ significantly between the pre-diabetic group and the longterm diabetic group of ZDF rats. Instead, there was significantly lower basal [35S]GTPγS binding in the spinal cord of ZDF rats at 29-wks c.f. 7-wks of age. Together, the findings suggest that impaired basal G-protein function rather than impaired coupling of MOP-r to its inhibitory G-protein may, at least in part, underpin -opioid agonist hyposensitivity in 29-wk ZDF rats. Finally, Chapter 7 contains a brief description of the main conclusions and discussion of the relevance of this doctoral research project, including potential future research directions.