Activin-A is a member of transforming growth factor-β (TGF-β) superfamily of cytokines which includes TGF-βs, Activins, Nodal, bone morphogenetic proteins (BMPs), growth and differentiation factors (GDFs) and anti-Mullerian hormone (AMH). TGF-β, Activin and Nodal are known to activate SMAD2/3, while BMPs and GDFs are known to activate SMAD1/5/8 signaling pathways. Activin-A binds to type II transmembrane serine threonine kinase receptor (ActRIIA or ActRIIB), which in turn activates type I receptor (ActRIB) leading to phosphorylation of SMAD2/SMAD3. Upon phosphorylation, SMAD2/3 forms a complex with SMAD4, which then translocates to nucleus. In the nucleus, SMAD2/3/4 complex, along with other co-factors regulates expression of a large number of genes. Unlike TGF-β, role of Activin in cancer is not well understood. Activin has been shown to be overexpressed in several cancers including metastatic prostate cancer, colorectal cancer, lung cancer, hepatocellular carcinoma and pancreatic cancer. Activin signaling has been shown to promote aggressiveness of esophageal squamous cell carcinoma and enhancing skin tumorigenesis and progression. Nodal, which binds to the same set of receptors, has also been shown to be overexpressed in several cancers. However, role of Activins in breast cancer progression is not well studied. Activin is expressed by normal breast epithelium and is known to play a role in mammary gland development. Earlier, a study had reported downregulation of Activin signaling in breast tumors. On the contrary, increased serum level of Activin has been reported in women with metastatic breast cancers. It is pertinent to mention here that TGF-β, which has been implicated in the progression and metastatic spread of breast cancers, also functions through the same set of downstream effectors- SMAD2 and SMAD3. Hence we wanted to evaluate the status of Activin signaling pathway in breast tumors and investigate its functional role in cancer progression. Gene expression profiling of 80 breast tumors and 20 normal samples was earlier performed in our laboratory revealed overexpression of INHBA in tumors compared to normal tissue samples. An independent set of 30 tumor and 15 normal samples were used to verify these results. Real-time PCR analysis revealed around 11.31 fold upregulation (p<0.001) of INHBA in breast tumors in comparison to normals. While no change in expression of INHA was observed, INHBB was found to be significantly downregulated in tumor samples. These results indicated upregulation of Activin-A in breast tumors. Further, a significant upregulation of ACVR2A and SMAD2 which act as signal transducers of Activin signaling pathway, was observed in breast tumors. Interestingly, while an increase in the expression of TGF-β1 was observed, TGFBR2 was found to be significantly downregulated in breast tumors. In addition, PCR analysis revealed significant downregulation of FST, β-glycan, IGSF1 and IGSF10, which act as negative regulators of Activin signaling pathway. Functional antagonism between TGF-β/Activin and BMP signaling pathway has been shown in both development and disease. Further analysis revealed that various BMPs including BMP2, BMP4 and BMP6 are downregulated in breast tumors compared to normal tissue samples. Various components and regulators of BMP signaling pathway were also found to be deregulated, indicating suppression of BMP signaling in breast tumors. To evaluate whether Activin signaling is active in breast tumor cells, immunohistochemistry with another set of 13 normal and 29 tumor samples was performed. Immunohistochemistry analysis revealed that most of the tumors have higher levels of Activin-A compared to normals tissues. Interestingly, no significant changes in expression of Activin-A was observed between normals and low grade tumors, suggesting that Activin-A may play an important role towards the late stages of the disease. In good correlation, breast tumors showed increased phospho SMAD2 and phospho SMAD3 levels compared to normal tissues. Also, in the same set of tumors, BMP2 staining showed a reduced expression pattern compared to normal tissues. Expression of inhibin in some normal and breast tumor samples revealed that most of the tumor samples have lower levels of inhibin compared to normal tissues. In order to understand the role of Activin-A in cancer progression, a panel of cell lines was selected. Treatment of cells with Activin-A resulted in activation of canonical SMAD as well as non-canonical Erk1/2 and PI3K signaling pathways. However, Activin-A treatment did not lead to activation of TAK1/p38 MAPK pathway. To begin with, it was important to evaluate effect of Activin-A on proliferation of various cell lines. Primarily, SMAD2/3 signaling pathway inhibits proliferation of normal epithelial cells, and hence, it is considered to have a tumor suppressive role. owever, this signaling pathway remains intact in most ( 98%) of the breast cancers. BrdU incorporation assay showed that Activin-A does not promote proliferation of cells under monolayer culture conditions. However, soft agar assay results showed that Activin signaling promotes anchorage independent growth of cancer cells. TGF-β is widely known as an inducer of epithelial mesenchymal transition (EMT). Also, EMT is considered to be a prerequisite for epithelial cells to undergo migration and invasion. During EMT, cells loose epithelial characteristics and acquire mesenchymal features along with cytoskeletal rearrangement. Treatment of cells with Activin-A resulted in downregulation of E-cadherin and upregulation of various mesenchymal markers. In addition, confocal microscopy imaging revealed a mesenchymal morphology of cells treated with Activin-A. Also, collagen gel contraction assay results indicated that Activin-A enhances the contractile property of HaCaT cells significantly. Cells undergone EMT are believed to acquire migratory and Invasive behaviour. In agreement with this, both scratch assay and trans-well migration assay showed that Activin-A enhances the migration of various cell lines. Further, Trans-well matrigel invasion assays were performed to assess how Activin affects invasion of various cancer cells. Matrigel invasion assay results showed that Activin-A enhances invasion of various cancer cell lines significantly. Also, RT-PCR, zymography and Luciferase assay results showed that Activin-A induces MMP2 expression. As described earlier, Activin-A activates both canonical as well as non canonical signaling pathways. In this direction, it was interesting to investigate the contribution of SMAD signaling pathway in pro-tumorigenic actions of Activin-A. Inhibiting SMAD3 activity either by its stable knockdown or by using a SMAD3 specific small molecule inhibitor revealed that Activin-A regulation of EMT markers is SMAD3 dependent. Further, it was observed that SMAD3 contributes significantly in mediating Activin-A induced migration and invasion. Hence, it is likely that SMADs may play an important role in breast tumor progression. Next, stable overexpression of Activin-A in MCF-7 or its knockdown in MDA-MB-231 and H460 cells was performed to assess the effect of Activin-A on the behaviour of these cells. BrdU assay indicated no change in proliferation of cells upon overexpression or knockdown of Activin-A. However, soft agar assay results showed that Activin-A expression affects anchorage independent growth of these cells. MCF-7 cells are generally considered to be less aggressive in their tumor forming ability. Activin-A overexpressing MCF7 cells and control cells were respectively injected into right and left flank of immunocompromised mice and followed till the tumors reached to a prominent size. Our results show that Activin-A overexpressing MCF-7 cells have better tumor forming ability in comparison to control cells. In contrast to MCF-7 cells, MDA-MB-231 cells are known to be aggressive in their tumorigenic potential. In order to understand the effect of Activin-A knockdown on the tumor forming ability in MDA-MB-231 cells, 0.5 million cells (optimal cell number generally used is 1-2 million) were injected subcutaneously in immunocompromised mice. The results showed that while control cells gave rise to a tumor in 7 out of 10 animals, Activin-A knockdown cells could form a tumor in only 3 out of 10 animals. Also, the tumors formed by control cells were significantly larger by weight as compared to tumors formed by knockdown cells. Further, immunohistochemistry showed that tumors formed by MCF-7 cells overexpressing Activin-A have higher Ki-67 percentage as compared to control tumors. One of the factors known to be important for tumor growth is VEGF, which leads to recruitment of blood vessels and hence providing nourishment to the tumor cells. Hence Activin-A regulation of VEGF expression was evaluated next. Activin-A treatment or its stable overexpression in MCF-7 cells resulted in increased VEGF expression in these cells. This was also confirmed by VEGF promoter activity assay. To assess if Activin-A can play a role in metastatic spread of cancer cells, tail vein injection of Activin-A overexpressing MCF-7 cells was performed in immunocompromised mice. Even though no significant difference was found in the number of nodules formed by control or Activin-A overexpressing cells, it was observed that Activin-A overexpressing cells formed much bigger nodules as compared to the control cells. This suggests that Activin-A may play an important part in the establishment of metastases from the disseminated cancer cells. Tumor forming ability of cancer cells and aggressiveness of various cancers has been associated with the presence of cells having stem-like phenotype. In this direction, CD44high and CD24low expression status was analysed upon overexpression and knockdown of Activin-A in MCF-7 and MDA-MB-231 cells respectively. FACS analysis of Activin-A overexpressing MCF-7 cells and Activin-A knockdown MDA-MB-231 cells shows that Activin-A expression leads to enrichment of breast cancer stem-like cells. In conclusion, this study highlights the importance of Activin-A signaling pathway in the progression of breast tumors. It is also important to note the role of SMAD signalling in the progression of breast cancers since these effectors are common between TGF-β, Activin and nodal factors, which have been shown to be involved in cancer progression in a context dependent manner.

No description available.

DCs are critical to the early events of HIV-1 infection. They are the first cells that HIV-1 encounters at mucosal surfaces and as sentinel antigen-presenting cells of the immune system these should alarm the immune system and activate innate immune defences to recruit effective adaptive immunity and viral clearance. A peculiar characteristic of HIV – in contrast to other ssRNA viruses – is its ability to completely evade host innate recognition pathways. Additionally, it has the unique ability to manipulate the endo-lysosomal system of DCs and promote transmission via trans-infection to CD4+ T cells across virological synapses. However, it is largely unknown how HIV-1 is sensed by the innate immune system. Here, a multipronged experimental approach based on phosphoproteomics, transcriptomics and custom RNAi screen was developed to characterize the early signaling complex induced by HIV-1 in DCs. A novel method of phosphoproteomics to identify the HIV-1 phosphoproteome in DCs showed that 342 proteins were differentially phosphorylated following 10 min of HIV-1 infection compared to time-matched mock-infected DCs. Functional analysis of these phosphoproteins showed enrichments in several cellular pathways, including vesicular trafficking, cytoskeletal rearrangements and the secretory pathway and a relative paucity of signaling molecules involved in inflammatory pathways. Proteomics analysis of HIV-1 virions was undertaken to identify host molecules hijacked by HIV-1 during viral replication and revealed a close interaction between the virus and the endo-lysosomal system. Transcriptomics analysis of HIV-1 infected DCs showed a muted immune response with no detectable differentially regulated genes. The results of the phoshoproteomic screen provided the basis for a custom RNAi screen to identify host proteins that are differentially phosphorylated by the virus and required for efficient trans-infection from DCs to CD4+ lymphocytes. The results of this screen showed that 54 of the 120 host factors tested were required for efficient viral transfer to CD4+ T cells and characterize the compartment that HIV-1 is internalized in on a molecular level. Two host factors identified within the HIV-1 phosphoproteome were chosen for further studies. Studies of BLOC-1 (biogenesis of lysosome-related organelles complex-1) and its subunits identified a role for snapin in HIV-1 trans-infection and HIV-1 and TLR8 sensing. Snapin may act as determinant of sorting of HIV-1 intraluminal vesicles to non-degradative, non-immunogenic compartments by activating mammalian target of rapamycin, mTOR, and inhibiting autophagy. Furthermore, HIV-1 triggered dephosphorylation of the cytosolic tyrosine phosphatase possibly via the interaction of host CD47 incorporated in the virion and the transmembrane glycoprotein SIRPα expressed on DCs. Blocking of this interaction with an inhibitory CD47 antibody resulted in a reduction of HIV-1 replication. Taken together, this multipronged approach reveals the complexity of the interaction of HIV-1 with the host cell machinery and identifies novel mechanism of the immune evasion tactics usurped by HIV-1.

No description available.

In the U.K. deaths due to liver diseases, especially alcohol related diseases, have risen considerably over the last 20 years. In 2005 up to 13,000 people died from liver related diseases within the U.K., including alcohol and viral liver failure and liver cancers. Worldwide hepatitis B affects about 2 billion people, killing 500,000 to 1 million per year. An effective way to treat liver disease is often liver surgery, such as liver resection for cancers and liver transplant for failure. However, the failure of liver regeneration by hepatocyte proliferation after resection surgery leads to a high death rate, and a shortage of liver donors means most people with liver failure die without access to a transplant. Therefore, understanding hepatocyte proliferation is a key to improving survival after resection surgery and providing hepatocytes for cell therapy in place of organ donation. The mechanism of hepatocyte proliferation has been studied both in vivo and in culture by many groups. However, only limited proliferation and preservation of function of primary human and rat hepatocytes, not suitable for clinical use, has been achieved on stimulation with growth factors. This study focuses on the mechanism of epidermal growth factor (EGF) stimulation of rat hepatocyte cell cycle progression and proliferation, including the role of PI3K/Akt/mTOR and MEK/ERK signalling pathways, EGF receptor location after activation of downstream proteins such as protein kinase B (Akt) and extracellular signal-regulated kinases 1/2 (ERK1/2), and their effect on the cell cycle. Included in this study are some comparisons between the stimulation of the EGF receptor (a tyrosine kinase receptor) and the P2Y receptor (a G protein coupled receptor). The PI3K/Akt/mTOR signalling pathway appears to be necessary for the hepatocyte response to EGF, inducing progression to S phase and DNA synthesis, while the MEK/ERK pathway is important but not necessary. The P2Y2 agonist UTP, which also stimulates these two pathways, does not result in the cell entering S phase. This suggests that the activation of these two signalling pathways is not sufficient for cell cycle progression. Furthermore, infection of cells with adenovirus to express constitutively active Akt increases hepatocytes proliferation and induces cell cycle progression, which generates a window to obtain hepatocytes proliferation in culture. It has been shown in this thesis that EGF stimulation of ERK phosphorylation continues from endosomes, while the evidence suggests that UTP stimulation is restricted to signalling at the cell surface. Furthermore, endocytic EGF/EGFR alone (without stimulation from the cell surface) is sufficient to induce cell cycle progression. This endosomal signalling with EGF but not UTP may explain the absence of cell cycle progression following UTP. EGF stimulates the appearance of phospho-EGFR in the nucleus. Furthermore, nuclear EGFR has a different apparent molecule weight than the cytoplasmic receptor; this may be due to nuclear EGFR having fewer and/or different phosphates. In vivo work by others has shown that in liver regeneration following partial hepatectomy (PH) EGF and full-length activated-EGFR were showed to be present in proliferating hepatocytes. This thesis describes the mechanism of growth factor (EGF) stimulation of primary rat hepatocyte proliferation. It shows for the first time that endosomal EGF/EGFR alone is sufficient to stimulate cell cycle progression, and that EGF induces phospho-EGFR in the nucleus in cultured rat hepatocytes. This thesis also provides the possibility to obtain cultured hepatocytes proliferation including over-expression of constitutively active form of Akt and translocation to the nucleus of full-length EGFR in the phosphorylated form. These studies improve our understanding of growth factor (e.g. EGF) stimulation of hepatocyte proliferation in vitro and help to move closer to the goal of obtaining sufficient functional hepatocytes in culture for clinical use, and of drugs that will stimulate hepatocyte proliferation following resection surgery.

<p>Much effort today in the Internet research community isaimed at providing network services for applications that werenot under consideration when the Internet was originallydesigned. Nowadays the network has to support real-timecommunication services that allow clients to transportinformation with expectations on network performance in termsof loss rate, maximum end-to-end delay, and maximum delayjitter. Today there exist two quality of service (QoS)architecture for the Internet: The integrated services, whichis usually referred to as intserv, and the differentiatedservices referred to as diffserv. Although the intserv clearlydefines the quality levels for each of its three serviceclasses, the limited scalability of this QoS architecture is acontinuous topic for discussion among the researchers. Theanalysis of the tradeoffs of the two QoS architecturesmotivated us to design a new QoS architecture which will takethe strength of the existing approaches and will combine themin a simpler, efficient and more scalable manner.</p><p>In this LicentiateThesis we introduce a guaranteed servicefor the Internet, which definition is similar to the one inintserv: The guaranteed service (GS) is a network servicerecommended for applications with firm requirements on qualityof end-to-end communication. The service should provide zeropacket loss in routers and tightly bound the end-to-end delay.The capacity for a GS connection should be explicitly reservedin every router along a path of a connection. However, incontrary to intserv the necessary quality level will beprovided without per-flow scheduling in the core routers, whichis the major drawback of the intserv architecture. We use thediffserv principle of dealing with aggregates in the corenetwork since this approach is proven to be scalable andefficient.</p><p>The thesis considers two major building blocks of the newarchitecture: The packet scheduling and the signaling protocol.We have developed a special scheduling algorithm. Our formaland experimental analysis of its delay properties shows thatthe maximum end-to-end delay is acceptable for real-timecommunication. Moreover, our scheme provides a fair service tothe traffic of other service classes. In order to achieve thedesired QoS level, a sufficient amount of capacity should bereserved for the GS connections in all intermediate routersend-to-end. We have developed a both simple and robustsignaling protocol. The realization of our protocol shows thatrouters are able to process up to 700,000 signaling messagesper second without overloading the processor.</p>

Arsenical-induced carcinogenesis in human bladder has been established through epidemiological evidence, but unfortunately, no mode of action had been determined for this phenomenon. UROtsa cells, a normal, immortalized cell culture model of human urothelium does not form tumors when injected into immuno-compromised mice nor does it have anchorage-independent growth. UROtsa cells were shown to be malignantly transformed following low-level exposure to both arsenite [As(III)] and its more toxic metabolite, monomethylarsonous acid [MMA(III)] providing additional models for studying arsenical-induced carcinogenesis of the bladder. These transformed cell lines allow researchers the ability to investigate the process of urothelial tumorigenesis at multiple time points of arsenical exposure. In the studies discussed here in, environmentally relevant levels of As(III) and MMA(III) were chosen. UROtsa cells were exposed to As(III) and MMA(III) both acutely and chronically to begin investigations into signaling pathway alterations that can lead to carcinogenesis in the human bladder upon exposure to arsenicals. In acute studies, it was shown that As(III) and MMA(III) generate oxidative stress response in UROtsa at low, environmentally relevant levels. The ROS generated by MMA(III) led to an increased 8-oxo-dG formation after 30 min, supporting the importance of MMA(III) in damage caused in the bladder by arsenicals. Because ROS has been linked to MAPK signaling, it was shown that 50 nM MMA(III) and 1 µM As(III) induce MAPK signaling following acute exposures and this increase is dependent on the production of ROS.Next, it was necessary to begin to look at changes that occur during transformation of UROtsa with MMA(III). Chronic exposure to 50 nM MMA(III) constitutively increases the amounts of EGFR, activated Ras, and COX-2 protein in MSC cells. Chronic upregulation of COX-2 in MSC52 cells is due to increased levels of ROS. Phenotypic changes seen in MSC52 cells (hyperproliferation and anchorage independent growth) are dependent on the secondary generation of excess ROS in MSC52 cells. These data clearly present evidence supporting a role for ROS in both acute and chronic toxicities associated with low-level arsenical exposure, and gives evidence that ROS are important in cellular transformation following MMA(III) exposure.

Precise regulation of heterotrimeric G-protein signaling is important for maintaining proper cardiovascular system function. Indeed, G-protein signaling is frequently upregulated during cardiovascular disease suggesting that identifying mechanisms for inhibiting G-protein signaling may be an effective therapeutic strategy for the treatment and prevention of disease. The work presented in this thesis is directed at two RGS proteins, RGS2 and RGS5, the two highest expressing RGS proteins in VSMCs. Despite the large number of studies published on them, there is still much to be learned about the specific G-protein pathways that each RGS protein controls. Using genetic and molecular models, we set out to identify novel regulatory pathways controlling RGS2 and RGS5 function. We hypothesize that characterizing the determinants and regulation of RGS protein function will provide a better understanding of the signaling that occurs within VSMCs under both physiologic and pathophysiologic conditions. Our work presented in the first three studies of this thesis, describes novel regulatory pathways that are involved in regulating RGS2 protein function. We describe the production of RGS2 protein isoforms that are the result of alternative translational start site usage. Interestingly, the expression pattern of these proteins is controlled by the signaling status of the cell. In the second two studies, we identify a functional consequence of RGS2-interaction with the plasma membrane. We show that this is dependent on the interaction between the amphipathic α-helix and anionic phospholipids present in the plasma membrane. We further show that disruptions in this interaction, as occurs in the human population, can lead to reduced RGS2 function and thus potentially hypertension. Finally, our last study focuses on the function and regulation of RGS5, the single highest expressing RGS protein in VSMCs. We show that the regulation of RGS5 is dependent, similar to other VSMC-specific genes, on the activity of SRF and myocardin. However, interestingly, RGS5 expression is further controlled by the extent of DNA methylation that occurs in its proximal promoter. We show that this is an important regulator of RGS5 expression both in development as well as during disease, specifically in-stent restenosis.

As sessile organisms, coordination of development and reproduction in a dynamic, and often stressful, environment presents a particular challenge for plants. Rapid processing of internal and external cues by complex signal transduction pathways leads to stimulus-appropriate physiological responses on an organismal scale. In plants, myriad signaling pathways are mediated by calcium (Ca2+) signals, and it is thought that different stimuli elicit unique patterns of Ca2+ influx into cells (termed Ca2+ ‘signatures’) that encode information important for proper physiological responses. Encoding of information in the form of Ca2+ signatures requires that decoding elements be present in cells to direct downstream cellular processes. This role is filled by Ca2+-binding proteins that serve as Ca2+ sensors. Interestingly, plant genomes encode multiple expanded families of Ca2+ sensors not found in animal genomes. Among these, the calmodulin (CaM)-like proteins (CMLs) are represented by a 50 member family in Arabidopsis. On the basis of structural homology, CMLs are predicted to function like conserved CaM, however, little work has been done to address this question. Biochemical characterization of CML39 indicates that it possesses structural properties consistent with function as a Ca2+ sensor. Analysis of transgenic CML39 loss-of-function (cml39) mutants revealed that CML39 is important for proper seedling establishment in the absence of exogenous metabolisable carbon as cml39 seedlings entered a state of developmental arrest shortly after germination. cml39 mutants also exhibited a conditional ‘de-etiolated’ phenotype when grown in complete darkness and exaggerated hypocotyl elongation under a short-day light regime. Genetic data suggest that CML39 functions in signaling pathways downstream of light perception, and this idea is supported by the observation that CML39 iii is expressed in light-sensing tissues, and that subunit 5 of the COP9 signalosome, a protein critical for photomorphogenesis, was identified as a putative target of CML39. Collectively, results show that CML39 is Ca2+ sensor that serves a critical regulatory role during seedling establishment when sucrose is limited, and importantly, further underscore the pervasiveness of Ca2+ signaling in plant growth and development. / Thesis (Ph.D, Biology) -- Queen's University, 2013-05-29 18:16:25.769

There is considerable evidence that cigarette smoking is the primary etiology of chronic obstructive pulmonary disease (COPD), and that oxidative stress occurs in COPD with the family of tissue nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox) enzymes playing a significant role in lung pathogenesis. The purpose of this study was to determine the effects of cigarette smoke extract (CSE) on Nox signaling to epithelial sodium channels (ENaCs). Pre-treatment with diphenyleneiodonium (DPI), a pan-Nox inhibitor, prevented stimulatory effects of CSE on ENaC activity; open probability (Po) changed from 0.36 +/- 0.09 to 0.11 +/- 0.02; n=10, p=0.01 following CSE and DPI exposure. Likewise, Fulvene-5 (which inhibits Nox2 and Nox4 isoforms) decreased the number of ENaC per patch (from 2.75 +/- 0.25 to 1 +/- 0.5, n=9, p=0.002) and open probability (0.18 +/- 0.08 to 0.02 +/- 0.08, p=0.04). Cycloheximide chase assays show that CSE exposure prevented alpha-ENaC subunit degradation, whereas concurrent CSE exposure in the presence of Nox inhibitor, Fulvene 5, resulted in normal proteolytic degradation of alpha-ENaC protein in primary isolated lung cells. In vivo, co-instillation of CSE and Nox inhibitor promoted alveolar flooding in C57Bl6 mice compared to accelerated rates of fluid clearance observed in CSE alone instilled lungs. Real-time PCR indicates that mRNA levels of Nox2 were unaffected by CSE treatment while Nox4 transcript levels significantly increased 3.5 fold in response to CSE. Data indicate that CSE is an agonist of Nox4 enzymatic activity, and that CSE-mediated Nox4 plays an important role in altering lung ENaC activity.