An immune modulatory role for the fes proto-oncogene

The Fes protein tyrosine kinase is abundantly expressed in phagocytic immune cells, including tumor associated macrophages. Fes knockout mice (fes-/-) display enhanced sensitivity to LPS, and this was shown to be associated with increased NF-κB signaling and TNFα production from fes-/- macrophages. Interestingly, tumor onset in the mouse mammary tumor virus (MMTV-Neu) transgenic mouse model of breast cancer is significantly delayed in fes-/- mice, and this was associated with increased frequency of CD11b+ myeloid and CD3+ T cells in the premalignant mammary glands. Recent studies have also implicated Fes in cross-talk between MHC-I and the NF-κB and IRF-3 pathways in macrophages. Signal 3, the production of inflammatory cytokines and Type I interferons downstream of NF-κB and IRF-3 pathways in antigen presenting cells, is considered an important component of T-cell activation, after engagement of T cell receptor by MHC presented antigen (Signal 1) and co-receptors by their ligands (Signal 2). Using a lymphocytic choriomeningitis virus (LCMV) model of immune activation, I show that LPS stimulated fes-/- macrophages promote more robust activation of LCMV antigenspecific CD8+ T cells than wild type macrophages (fes+/+). Furthermore, LPS stimulated fes-/- macrophages showed increased phosphorylation of NF-B and IRF-3. I also showed that Fes colocalizes with MHC-I in dynamic vesicular structures within macrophages. These observations are consistent with a model where Fes regulates Signal 3 in antigen presenting cells through roles in cross-talk between MHC-I and the NF-kB and IRF-3 signaling pathways. This suggests that Fes plays an immune checkpoint role at the level of Signal 3, and that Fes inhibition could promote tumor immunity through increased Signal 3 driven T cell activation.

Identification and Development of Enzymes Associated With the Sperm Inner Acrosomal Membrane and Their Function during Fertilization

In order for mammalian fertilization to transpire, spermatozoa must transit through the female reproductive tract and penetrate the outer investments of the oocyte: the cumulus oophorus and the zona pellucida. In order to penetrate the oocyte, spermatozoa must undergo the acrosome reaction. The acrosome reaction results in the exposure of the inner acrosomal membrane (IAM) and proteins that coat it to the extracellular environment. After the acrosome reaction, the IAM becomes the leading edge of spermatozoa undergoing progressive movement. Thus the enzymes which effect lysis of the oocyte investments ought to be located on the IAM. An objective of this study was to identify and characterize enzymatic activity detected on the IAM and provide evidence that they play a role in fertilization. This study also describes procedures for fractionating spermatozoa and isolating the IAM and proteins on its intra- and extra-vesicular surfaces, and describes their development during male gametogenesis. Since the IAM is exposed to the extracellular environment and oviductal milieu after the acrosome reaction, this study also sought to characterize interactions and relationships between factors in the oviductal environment and the enzymes identified on the IAM. The data presented provide evidence that MMP2 and acrosin are co-localized on the IAM, originate from the Golgi apparatus in gametogenesis, and suggest they cooperate in their function. Their localization and results of in vitro fertilization suggests they have a function in zona pellucida penetration. The data also provide evidence that plasminogen, originating from the oviductal epithelium and/or cumulus-oocyte complex, is present in the immediate environment of sperm-egg initial contact and penetration. Additionally, plasminogen interacts with MMP2 and enhances its enzymatic action on the IAM. The data also provide evidence that MMP2 has an important function in penetration of the cumulus oophorus. Holistically, this thesis provides evidence that enzymes on the IAM, originating from the Golgi apparatus in development, have an important function in penetration of the outer investments of the oocyte, and are aided in penetration by plasminogen in the female reproductive tract.

Access Point Association Coordination in Densely Deployed 802.11 Wireless Networks

Dense deployment of wireless local area network (WLAN) access points (APs) is an important part of the next generation Wi-Fi and standardization (802.11ax) efforts are underway. Increasing demand for WLAN connectivity motivates such dense deployments, especially in geographical areas with large numbers of users, such as stadiums, large enterprises, multi-tenant buildings, and urban cities. Although densification of WLAN APs guarantees coverage, it is susceptible to increased interference and uncoordinated association of stations (STAs) to APs, which degrade network throughput. Therefore, to improve network throughput, algorithms are proposed in this thesis to optimally coordinate AP associations in the presence of interference. In essence, coordination of APs in dense WLANs (DWLANs) is achieved through coordination of STAs' associations with APs. While existing approaches suggest tuning of APs' beacon powers or using transmit power control (TPC) for association control, here, the signal-to-interference-plus-noise ratio (SINRs) of STAs and the clear channel assessment (CCA) threshold of the 802.11 MAC protocol are employed. The proposed algorithms in this thesis enhance throughput and minimize coverage holes inherent in cell breathing and TPC techniques by not altering the transmit powers of APs, which determine cell coverage. Besides uncoordinated AP associations, unnecessary frequent transmission deferment is envisaged as another problem in DWLANs due to the clear channel assessment aspect of the carrier sensing multiple access collision avoidance (CSMA/CA) scheme in 802.11 standards and the short spatial reuse distance between co-channel APs. To address this problem in addition to AP association coordination, an algorithm is proposed for CCA threshold adjustment in each AP cell, such that CCA threshold used in one cell mitigates transmission deferment in neighboring cells. Performance evaluation reveals that the proposed association optimization algorithms achieve significant gain in throughput when compared with the default strongest signal first (SSF) association scheme in the current 802.11 standard. Also, further gain in throughput is observed when the CCA threshold adjustment is combined with the optimized association. Results show that when STA-AP association is optimized and CCA threshold is adjusted in each cell, throughput improves. Finally, transmission delay and the number of packet re-transmissions due to collision and contention significantly decrease.

THE IMPACT OF VIRUS INFECTION IN DEREGULATION OF CYTOKINE PRODUCTION UPON SECONDARY BACTERIAL INFECTION

Dendritic cells (DCs) secrete cytokines such as interleukin-23 (IL-23) when stimulated with certain Toll-like receptor (TLR) agonists and infected with pathogens such as P. aeruginosa. IL- 23 is a proinflammatory cytokine that plays a critical role in the proliferation and differentiation of the IL-17 producing Th17- CD4 T helper cells. The lack of efficient cytokine production from antigen-presenting cells, such as DCs, can impact CD4 differentiation and thus impair the immune responses against pathogens. Clearance of some bacterial infections, such as Klebsiella pneumonia and Listeria monocytogenes has been shown to be dependent on the induction of IL-23 and therefore, deregulation of these cytokines as a direct result of virus infection may impede immune responses to secondary infections. Here, an inhibition of TLR ligand or P. aeruginosa-induced IL- 23 expression in Lymphocytic Choriomeningitis Virus (LCMV)-infected bone marrow-derived dendritic cells (BMDCs) has been demonstrated, indicating that an important function of these cells is disrupted during virus/bacterial coinfection. While production of TNF-α was unaffected in LPS stimulated cells, TNF-α was significantly inhibited in bacterium infected cells by LCMV. Type I IFN in LPS or LCMV infected cell was not detected and therefore, ruling out the possibility of cytokine suppression by Type I IFN. The production of IL-10 was high in BMDCs infected with LCMV and stimulated with LPS or bacteria. Analysis of multiple cytokines produced in this coinfection model demonstrated that LCMV infection impacts specific cytokine production upon LPS or bacterium infection, which may be important for bacterial clearance. This data is important for future immunotherapy use in viral/bacterial coinfection scenarios.

Relationship between abnormal maternal inflammation and insulin resistance during pregnancy

Abnormal maternal inflammation during pregnancy is linked to complications such as preeclampsia and fetal growth restriction. There is growing evidence that insulin resistance is also associated with a heightened inflammatory state, and is linked to pregnancy complications such as gestational diabetes. This study tested the hypothesis that abnormal inflammation during pregnancy is causally linked to elevations in blood glucose and insulin resistance. To induce a state of abnormal systemic inflammation, bacterial lipopolysaccharide (LPS) was administered to pregnant rats on gestational days (GD) 13.5-16.5. Dams treated with LPS exhibited an abnormal immune response characterized by an elevation in white blood cells, which was linked to reduced fetal weight and increased glucose levels over pregnancy. Abnormal inflammation is characterized by increased levels of circulating pro-inflammatory cytokines such as tumour necrosis factor alpha (TNF) and interleukin-6, which contribute to insulin resistance by inhibiting the insulin signalling pathway. TNF in particular induces a serine phosphorylation (pSer307) of insulin receptor substrate 1 (IRS-1). In our model, insulin resistance was assessed by measuring the extent of pSer307 of IRS-1 and total IRS-1 expression in skeletal muscle, as well as changes in metabolic parameters and pancreas tissue morphology associated with insulin resistance. LPS-treated dams exhibited a significant reduction in IRS-1 expression, elevation in fasting glucose levels, and reduction in insulin sensitivity indices. There were also biologically relevant increases in fasting plasma insulin levels and insulin resistance indices, but not pSer307 of IRS-1 and pancreatic islet size. To determine whether inflammation plays a role in reducing insulin signalling and the other changes associated with LPS administration, etanercept, a TNF antagonist, was administered on GDs 13.5 and 15.5 prior to LPS injections. With the exception of IRS-1 expression, in rats treated with etanercept all of the measured parameters remained at the levels observed in saline controls, indicating a link between abnormal inflammation and insulin resistance. The results of this study support the practice of monitoring the inflammatory conditions of the mother prior to and during pregnancy, and support further investigation into the potential use of anti-inflammatory agents during pregnancy in women at risk of insulin resistance and gestational diabetes.

Optimizing The Nasal Allergen Challenge Model For The Study Of Allergic Rhinitis

Background The Allergic Rhinitis Clinical Investigator Collaborative (AR-CIC) uses a Nasal Allergen Challenge (NAC) model to study the pathophysiology of AR and provides proof of concept for novel therapeutics. The NAC model needs to ensure optimal participant qualification, allergen challenge, clinical symptoms capture and biological samples collection. Repeatability of the protocol is key to ensuring unbiased efficacy analysis of novel therapeutics. The effect of allergen challenge on IL-33 gene expression and its relation to IL1RL1 receptor and cytokine secretion was investigated. Methods Several iterations of the NAC protocol was tested, comparing variations of qualifying criteria based on the Total Nasal Symptom Score (TNSS) and Peak Nasal Inspiratory Flow (PNIF). The lowest allergen concentration was delivered and TNSS and PNIF recorded 15 minutes later. Participants qualified if the particular criteria for the protocol were met, otherwise the next higher allergen concentration (4-fold increase), was administered until the targets were reached. Participants returned for a NAC visit and received varying allergen challenge concentrations depending on the protocol, TNSS/PNIF were recorded at 15 minutes, 30 minutes, 1 hour, and hourly up to 12 hours, a 24 hour time point was added in later iterations. Repeatability was evaluated using a 3-4week interval between screening, NAC1, and NAC2 visits. Various biomarker samples were collected. Results A combined TNSS and PNIF criterion was more successful in qualifying participants. The cumulative allergen challenge (CAC) protocol proved more reliable in producing a robust clinical and biomarker response. Repeatability of the CAC protocol was achieved with a 3-week interval between visits, on a clinical and biological basis. IL-33 cytokine is an important biomarker in initiating the inflammatory response in AR in humans. IL-33 and IL1RL1 expression might employ a negative feedback mechanism in human nasal epithelial cells. Comparing the clinical and biological response to ragweed vs cat allergen challenge, proved the CAC protocol’s suitability for use employing different allergens. Conclusion The AR-CIC’s CAC protocol is an effective method of studying AR, capable of generating measurable and repeatable clinical and biomarker responses, enabling better understanding of AR pathophysiology and ensuring that any change would be purely due to medication under investigation in a clinical trial setting.

Differences in Abdominal and Liver Fat Accumulation Between Obesity-Matched Adults With and Without Type 2 Diabetes

Despite attempts to identify the mechanisms by which obesity leads to the development of Type 2 Diabetes (T2D), it remains unclear why some but not all adults with obesity develop T2D. Given the established associations between visceral adipose tissue (VAT) and liver fat with insulin resistance, we hypothesized that compared to age and obesity matched adults who were non-diabetic (NT2D), adults with T2D would have greater amounts of VAT and liver fat. The International Study of Prediction of Intra-Abdominal Adiposity and Its Relationship with Cardiometabolic Risk/Intra-Abdominal Adiposity (INSPIRE ME IAA) aims to study the associations between VAT and liver fat and risk of developing T2D and cardiovascular disease. Four thousand, five hundred and four participants were initially recruited; from this, 2383 White and Asian adults were selected for this ancillary analysis. The NT2D and T2D groups were matched for age, body mass index (BMI) and waist circumference (WC). The T2D and NT2D groups were also compared to participants with either impaired fasting glucose (IFG) or impaired glucose tolerance (IGT; IFG/IGT)). Abdominal adipose tissue was measured by computed tomography; liver fat was estimated using computed tomography-derived mean attenuation. Secondary analysis determined whether differences existed between NT2D and T2D groups in VAT and liver fat accumulation within selected BMI categories for Whites and Asians. We report across sex and race, T2D and IFG/IGT groups had elevated VAT and liver fat compared to the NT2D group (p<0.05). VAT was not different between IFG/IGT and T2D groups (p>0.05), however liver fat was greater in the T2D group compared to the IFG/IGT group in both Whites and Asians (p<0.05). Within each BMI category, the T2D group had elevated VAT and liver fat compared to the age and anthropometrically matched NT2D group in both Whites and Asians (p<0.05). With few exceptions, abdominal subcutaneous adipose tissue was not different in the T2D or IFG/IGT groups compared to the NT2D group independent of sex and race. Compared to age and obesity-matched adults who are NT2D, we observe that White and Asian adults with T2D, and those with IFG/IGT, present with greater levels of both VAT and liver fat.

The Function of Intestinal Afferent Neurons in Regulating Satiety During Health and Obesity

Background and aim: Within the gastrointestinal tract, vagal afferents regulate satiety and food intake via chemical and mechanical mechanisms. Cysteinyl Leukotrienes (CysLTs) are lipid mediators that are believed to regulate food intake and body weight. However, the involvement of vagal afferents in this effect remains to be established. Conversely, Glucagon like peptide-1 (GLP-1) is a satiety and incretin peptide hormone. The effect of obesity on GLP-1 mediated gut-brain signaling has yet to be investigated. Since intestinal vagal afferents’ activity is reduced during obesity, it is intriguing to investigate their responses to GLP-1 in such conditions. Methods: Extracellular recordings were performed on intestinal afferents from normal C57Bl6, low fat fed (LFF), and high fat fed (HFF) mice. To examine the effect on neuronal calcium signaling, calcium-imaging experiments were performed on isolated nodose ganglion neurons. Food intake experiments were conducted using LFF and HFF mice. Oral glucose tolerance tests (OGTT) were carried out. Whole cell patch clamp recordings were performed on nodose ganglion neurons from A) normal C57Bl mice to test the effect of CysLTs on membrane excitability, B) LFF and HFF mice to examine GLP-1 effect on membrane excitability during obesity. c-Fos immunohistochemical techniques were performed to measure the level of neuronal activation in the brainstem of both LFF and HFF mice in response to Ex-4. Results: CysLTs increased intestinal afferent firing rate and mechanosensitivity. In single nodose neuron experiments, CysLTs increased excitability. The GLP-1 agonist Ex-4 significantly decreased food intake in LFF but not HFF mice. However, Ex-4 markedly attenuated the rise in blood glucose in both LFF and HFF mice. The observed increase in nerve firing and mechanosensitivity following the application of GLP-1 and Ex-4 was abolished in HFF mice. Cell membrane excitability was significantly increased by Ex-4 in nodose from LFF but not HFF mice. Ex-4 significantly increased the number of activated neurons in the NTS area of LFF mice but not in their HFF counterparts. Conclusion: The previous observations indicate that the role CysLTs play in regulating satiety is likely to be vagally mediated. Also that satiety, but not incretin, effects of GLP-1 are impaired during obesity.

Hierarchical synthesis of complex DSP functions on FPGAs

SoC systems are now being increasingly constructed using a hierarchy of subsystems or silicon Intellectual Property (IP) cores. The key challenge is to use these cores in a highly efficient manner which can be difficult as the internal core structure may not be known. A design methodology based on synthesizing hierarchical circuit descriptions is presented. The paper employs the MARS synthesis scheduling algorithm within the existing IRIS synthesis flow and details how it can be enhanced to allow for design exploration of IP cores. It is shown that by accessing parameterised expressions for the datapath latencies in the cores, highly efficient FPGA solutions can be achieved. Hardware sharing at both the hierarchical and flattened levels is explored for a normalized lattice filter and results are presented.

Linear QR architecture for a single chip adaptive beamformer

This paper presents the design of a novel single chip adaptive beamformer capable of performing 50 Gflops, (Giga-floating-point operations/second). The core processor is a QR array implemented on a fully efficient linear systolic architecture, derived using a mapping that allows individual processors for boundary and internal cell operations. In addition, the paper highlights a number of rapid design techniques that have been used to realise this system. These include an architecture synthesis tool for quickly developing the circuit architecture and the utilisation of a library of parameterisable silicon intellectual property (IP) cores, to rapidly develop detailed silicon designs.

Bad Faith in European Trade Mark Law

Analyses how the European Court of Justice has interpreted the EU law rules against the registration of a trade mark or Community trade mark by an applicant in bad faith. Reviews case law from the UK courts, Office of Harmonisation in the Internal Market and Community courts on the role of bad faith as a moral standard. Considers case law on the narrow interpretation of bad faith in view of other EU provisions limiting trade marks.