The expression and regulation of pregnancy-specific glycoproteins in the mouse

Pregnancy-Specific Glycoproteins (PSG) are the most abundant fetally expressed proteins in the maternal bloodstream at term. This multigene family are immunoglobulin superfamily members and are predominantly expressed in the syncytiotrophoblast of human placenta and in giant cells and spongiotrophoblast of rodent placenta. PSGs are encoded by seventeen genes in the mouse and ten genes in the human. Little is known about the function of this gene family, although they have been implicated in immune modulation and angiogenesis through the induction of cytokines such as IL-10 and TGFβ1 in monocytes, and more recently, have been shown to inhibit the platelet-fibrinogen interaction. I provide new information concerning the evolution of the murine Psg genomic locus structure and organisation, through the discovery of a recent gene inversion event of Psg22 within the major murine Psg cluster. In addition to this, I have performed an examination of the expression patterns of individual Psg genes in placental and non-placental tissues. This study centres on Psg22, which is the most abundant murine Psg transcript detected in the first half of pregnancy. A novel alternative splice variant transcript of Psg22 lacking the protein N1-domain was discovered, and similar to the full length isoform induces TGFβ1 in macrophage and monocytic cell lines. The identification of a bidirectional antisense long non-coding RNA transcript directly adjacent to Psg22 and its associated active local chromatin conformation, suggests an interesting epigenetic gene-specific regulatory mechanism that may be responsible for the high level of Psg22 expression relative to the other Psg family members upon trophoblast giant cell differentiation

Role of KLF4 in regulation of myocardin induced SMC differentiation in human smooth muscle stem progenitor cells (hSMSPC)

The differentiation of stem cells into multiple lineages has been explored in vascular regenerative medicine. However, in the case of smooth muscle cells (SMC), issues exist concerning inefficient rates of differentiation. In stem cells, multiple repressors potentially downregulate myocardin, the potent SRF coactivator induced SMC transcription including Krüppel like zinc finger transcription factor-4 (KLF4). This thesis aimed to explore the role of KLF4 in the regulation of myocardin gene expression in human smooth muscle stem/progenitor cells (hSMSPC), a novel circulating stem cell identified in our laboratory which expresses low levels of myocardin and higher levels of KLF4. hSMSPC cells cultured in SmGM2 1% FBS with TGF-β1 (5 ng/ml “differentiation media”) show limited SMC cell differentiation potential. Furthermore, myocardin transduced hSMSPC cells cultured in differentiation media induced myofilamentous SMC like cells with expression of SM markers. Five potential KLF4 binding sites were identified in silico within 3.9Kb upstream of the translational start site of the human myocardin promoter. Chromatin immunoprecipitation assays verified that endogenous KLF4 binds the human myocardin promoter at -3702bp with Respect to the translation start site (-1). Transduction of lentiviral vectors encoding either myocardin cDNA (LV_myocardin) or KLF4 targeting shRNA (LV_shKLF4 B) induced human myocardin promoter activity in hSMSPCs. Silencing of KLF4 expression in differentiation media induced smooth muscle like morphology by day 5 in culture and increased overtime with expression of SMC markers in hSMSPCs. Implantation of silastic tubes into the rat peritoneal cavity induces formation of a tissue capsule structure which may be used as vascular grafts. Rat SMSPCs integrate into, strengthen and enhance the SMC component of such tubular capsules. These data demonstrate that KLF4 directly represses myocardin gene expression in hSMSPCs, which when differentiated, provide a potential source of SMCs in the development of autologous vascular grafts in regenerative medicine.

Investigation of toll-like receptor tolerance in the regulation of inflammation

Inflammation is a complex and highly organised immune response to microbes and tissue injury. Recognition of noxious stimuli by pathogen recognition receptor families including Toll-like receptors results in the expression of hundreds of genes that encode cytokines, chemokines, antimicrobials and regulators of inflammation. Regulation of TLR activation responses is controlled by TLR tolerance which induces a global change in the cellular transcriptional expression profile resulting in gene specific suppression and induction of transcription. In this thesis the plasticity of TLR receptor tolerance is investigated using an in vivo, transcriptomics and functional approach to determine the plasticity of TLR tolerance in the regulation of inflammation. Firstly, using mice deficient in the negative regulator of TLR gene transcription, Bcl-3 (Bcl-3-/-) in a model of intestinal inflammation, we investigated the role of Bcl-3 in the regulation of intestinal inflammatory responses. Our data revealed a novel role for Bcl-3 in the regulation of epithelial cell proliferation and regeneration during intestinal inflammation. Furthermore this data revealed that increased Bcl-3 expression contributes to the development of inflammatory bowel disease (IBD). Secondly, we demonstrate that lipopolysaccharide tolerance is transient and recovery from LPS tolerance results in polarisation of macrophages to a previously un-described hybrid state (RM). In addition, we identified that RM cells have a unique transcriptional profile with suppression and induction of genes specific to this polarisation state. Furthermore, using a functional approach to characterise the outcomes of TLR tolerance plasticity, we demonstrate that cytokine transcription is uncoupled from cytokine secretion in macrophages following recovery from LPS tolerance. Here we demonstrate a novel mechanism of regulation of TLR tolerance through suppression of cytokine secretion in macrophages. We show that TNF-α is alternatively trafficked towards a degradative intracellular compartment. These studies demonstrate that TLR tolerance is a complex immunological response with the plasticity of this state playing an important role in the regulation of inflammation.

Legal regulation of the software "patentable subject matter" requirement in the US and in Europe: The need for certainty, predictability and uniformity

This thesis critically investigates the divergent international approaches to the legal regulation of the patentability of computer software inventions, with a view to identifying the reforms necessary for a certain, predictable and uniform inter-jurisdictional system of protection. Through a critical analysis of the traditional and contemporary US and European regulatory frameworks of protection for computer software inventions, this thesis demonstrates the confusion and legal uncertainty resulting from ill-defined patent laws and inconsistent patent practices as to the scope of the “patentable subject matter” requirement, further compounded by substantial flaws in the structural configuration of the decision-making procedures within which the patent systems operate. This damaging combination prevents the operation of an accessible and effective Intellectual Property (IP) legal framework of protection for computer software inventions, capable of securing adequate economic returns for inventors whilst preserving the necessary scope for innovation and competition in the field, to the ultimate benefit of society. In exploring the substantive and structural deficiencies in the European and US regulatory frameworks, this thesis develops to ultimately highlight that the best approach to the reform of the legal regulation of software patentability is two-tiered. It demonstrates that any reform to achieve international legal harmony first requires the legislature to individually clarify (Europe) or restate (US) the long-standing inadequate rules governing the scope of software “patentable subject matter”, together with the reorganisation of the unworkable structural configuration of the decision-making procedures. Informed by the critical analysis of the evolution of the “patentable subject matter” requirement for computer software in the US, this thesis particularly considers the potential of the reforms of the European patent system currently underway, to bring about certainty, predictability and uniformity in the legal treatment of computer software inventions.

The role played by angiotensin (1-7) in the regulation of renal haemodynamics and excretory function in anesthetized rats

Initial studies have demonstrated that intra- renal infusion of Ang (1-7) caused a diuresis and natriuresis that was proportional to the degree of activation of the Renin Angiotensin Aldosterone System (RAAS). This raised the question as why the magnitude of this diuresis and natriuresis was compromised in rats receiving a high sodium diet (suppressed RAAS) and enhanced in low sodium fed rats (activated RAAS)? Could the answer lie with changes in intra-renal AT1 or Mas receptor expression? Interestingly, the observed Ang (1-7) induced increases in sodium and water excretion in rats receiving either a low or normal sodium diet were and blocked in the presence of the AT 1 receptor antagonist (Losartan) in the presence of the, 'Mas' receptor antagonist (A-779). These data suggest that both AT1 and 'Mas' receptors need to be functional in order to fully mediate the renal responses to intra-renal Ang (1-7) infusion. Importantly, further experimentation also revealed that there is a proportional relationship between AT 1 receptor expression in the rat renal cortex and the magnitude of the excretory actions of intra renal Ang (1-7) infusion, which is only partially dependent on the level of 'Mas' receptor expression. These observations suggest that although Ang (1-7) induced increases in sodium and water excretion are mediated by the Mas receptor, the magnitude of these excretory responses appear to be dependent upon the level of AT 1 receptor expression and more specifically Ang II/ AT 1 receptor signalling. Thus in rats receiving a low sodium diet, Ang (1-7) acts via the Mas receptor to inhibit Ang II/ AT 1 receptor signalling. In rats receiving a high sodium diet the down regulated AT 1 receptor expression implies a reduction in Ang II/ AT 1 receptor signalling which renders the counter-regulatory effects of intra-renal Ang (1-7) infusion redundant.

The evolution of the medical professions in eighteenth-century Ireland: An institutional perspective

Ireland, in the eighteenth century, followed the classic tripartite division of regular medical practitioners into physicians, surgeons and apothecaries. At the beginning of the century surgeons and apothecaries were regarded as mere tradesmen, but by the end of the century both were regarded as professionals and had the right to regulate their respective professions. Practitioners in different regions of Europe developed in a different manner, and eighteenth-century practitioners in Ireland developed independently from their English counterparts. In common with Britain and Europe in the eighteenth century, the total number of practitioners increased in Ireland, and by the end of the century, apothecaries were the largest group in Dublin, closely followed by the surgeons. Surgeons and apothecaries at the start of the eighteenth century belonged to the same guild. However in mid-century, St Luke's guild of apothecaries was established and this provided the apothecaries with a new identity that allowed them to pursue auto regulation, rather than hitherto, when they had been regulated by the physicians. This was vital to the apothecaries as they were in direct commercial competition with both the physicians and the surgeons and faced increasing pressure from both druggists and the disparate group of practitioners known as the irregulars. The 1765 County Infirmaries Act established a hospital in virtually every county in Ireland, and cast the surgeon as the primary medical officer in the countrywide network of hospitals. This legislation, which was unique in Europe, had the unintended consequence of elevating the status of the surgeons, as prior to this physicians were always in the ascendancy in the voluntary hospitals in Ireland and Britain, in contrast to France. The status of the surgeons was further enhanced by the establishment of the College of Surgeons in Ireland in 1784, which provided them with a new corporate identity, the authority to regulate the profession countrywide, and, also, the ability to educate surgeons in Ireland. The establishment of the College of Surgeons placed further pressure on the apothecaries to demonstrate that they also had a recognisable identity, and the authority to regulate their own profession. This was achieved with the 1791 Apothecaries Act which established the Apothecaries Hall and give the apothecaries the right to regulate themselves. This innovative legislation deemed the apothecaries a profession, and was enacted twenty-four years prior to similar legislation in Britain. Commercial pressure from druggists and, probably, irregulars expedited the requirement of the apothecaries to establish a new corporate identity, in order to distance themselves from these groups. The changing status of both apothecaries and surgeons had little effect on the physicians as a group, and, despite being the beneficiaries of a generous bequest from Sir Patrick Dun in 1711 to provide medical chairs in Dublin, the physicians displayed an inertia during the eighteenth century that was not in keeping with the developments that occurred in the contemporary Dublin medical world. The fact that it took ninety-five years, and that five acts of parliament, two House of Commons enquiries and a House of Lords enquiry were required to ensure that Dun's wishes were brought to fruition demonstrates that the physicians did not develop at the same pace as the other medical groups in the city. Had Dun’s bequest been implemented as he desired, Dublin, with a number of voluntary hospitals, would have been well placed to provide comprehensive tuition for medical students in the eighteenth century. It was not until the nineteenth century that the city, and the populace, benefited from this legacy. This thesis will trace these developments in the context of changes that occurred in contemporary medical education and diagnosis in Ireland, Britain and France. It will demonstrate that Irish practitioners developed independently, influenced mainly by local issues, but also by those who had travelled abroad and returned to Ireland with new concepts and ideas, ensuring that Irish medical practitioners had the institutional structure that could encompass the diagnostic and regulatory changes that would become accepted in the nineteenth century.

Investigation of lantibiotic regulation, immunity and synergy

Due to the increasing incidence of antibiotic resistant strains, the use of novel antimicrobials, such as bacteriocins, has become an ever more likely prospect. Lacticin 3147 (of which there are two components, Ltnα and Ltnβ) and nisin belong to the subgroup of bacteriocins called the lantibiotics, which has attracted much attention in recent years. The lantibiotics are antimicrobial peptides that contain unusual amino acids resulting from a series of enzyme-mediated post translational modifications. Given that there have been relatively few examples of lantibiotic-specific resistance; these antimicrobials appear to represent valid alternatives to classical antibiotics. However, the fact that lantibiotics are naturally only produced in small amounts often hinders their commercialisation. In order to overcome this bottleneck, several approaches can be employed. For example, we can create a situation that reduces the quantity of a lantibiotic required to inhibit a target by combining it with other antimicrobials. Here, following an initial screen involving lacticin 3147 and several classical antibiotics, it was observed between lacticin 3147 and the commercial antibiotics polymyxin B/E function synergistically. This reduced the amounts of the individual antimicrobials required for kill and broadened the spectrum of inhibition of both agents. Upon combination with polymyxins, lacticin 3147, which has been associated with Gram positive targets only, actively targeted Gram negative species such as Escherichia coli and Cronobacter sp. An alternative means of addressing problems associated with lantibiotic yield is to better understand how production is regulated, and ultimately use this information to enhance peptide levels. With this in mind the regulation of lacticin 3147 production from the promoter Pbac was investigated using a green fluorescent protein (GFP) expression reporter system. This revealed that elements within both of the divergent operons of the lacticin 3147 gene cluster are involved in Pbac regulation. That is, LtnR, already established as a negative regulator of itself and the lacticin 3147 associated immunity genes, also acts as an activator of Pbac transcription. In contrast, an enhanced level of expression is observed in the absence of the lacticin 3147 structural genes, ltnA1 and ltnA2, indicating that these genes/gene products are involved in Pbac repression. In fact, through complementation of the ltnA2 gene, it was revealed that this regulation is more likely to be dependent on the presence of the gene transcript rather that the corresponding prepropeptide or modified Ltnβ. It may be that if lacticin 3147 production is successfully enhanced, the ability of the producing cell to protect itself may become an issue. To prepare for such a possibility a bioengineered derivative of the lacticin 3147 immunity protein LtnI (LtnI I81V) which provides enhanced protection was discovered through an in depth investigation involving the site and saturation mutagenesis of this protein. In addition, the creation of truncated forms of LtnI allowed the identification of important and essential regions of this immunity protein. Finally, as mentioned, self-immunity is essential to prevent self-killing. However the discovery of nisin U immunity and regulatory gene homologues (spiFEGRR’K) within the pathogenic strain S. infantarius subsp. infantarius is a cause for concern as it represents an example of immune mimicry, a form of lantibiotic-specific resistance. The ability of spiFEG to confer protection was apparent when they successfully provided protection to nisin A, F, Z, Q and U when expressed heterologously in the nisin sensitive L. lactis HP host. As a consequence of the studies presented in this thesis, it is likely that strategies will emerge that will facilitate the production of greater levels of lacticin 3147 production and lead to enhanced immunity in lactococcal backgrounds. Alternatively the need for enhanced production could be avoided through the use of antimicrobial combinations. In addition, providing awareness of the threats of the emergence of resistance through immune mimicry can allow researchers to develop strategies to prevent this phenomenon from leading to the dissemination of lantibiotic resistance.

A study of the regulation of Trib family members expression in normal and malignant haematopoiesis

The Tribbles family of genes consist of three members; TRIB1, TRIB2 and TRIB3. Trib1 and Trib2 have been identified as oncogenes that can induce AML in mice. However little is known about how the expressions of the Tribbles family genes are controlled in the cell during haematopoiesis or leukaemogenesis. To investigate the Tribbles genes in leukaemia a bioinformatics approach was used. TRIB2 expression was found to be elevated in T-ALL and ALL with t(1;19). TRIB1 was found not to be significantly elevated in any leukaemic subtypes. Analyses of the TRIB1 and TRIB2 gene signatures in both leukaemic and normal haematopoietic cells identified pathways and transcription factors associated with these signatures. Pathways enriched for the TRIB1 signature included TLR signalling pathways and NF-κB pathways. Transcription factors enriched for this signature include C/EBP and SRF. Enriched for the TRIB2 signature includes T cell signalling pathways and Notch signalling pathways. Transcription factors enriched for this signature include E2F and ETS. Further investigation in vitro confirmed the finding that E2F1 was as a potential regulator of TRIB2 expression. E2F1 is able to directly bind to the TRIB2 promoter region and induce TRIB2 expression. C/EBPα p42 was found to inhibit E2F1 and the p30 isoform was found to cooperate with E2F1 induced activation of the TRIB2 promoter. Indicating the potential presence of a regulatory loop involved in the regulation of the TRIB2 gene. In conclusion we have investigated the Tribbles gene signatures in both normal haematopoietic and leukaemic cells. This has led to the identification of a number of pathways and transcription factors associated with these genes. We have also identified a family of transcription factors directly responsible for the regulation of TRIB2 expression. This regulatory pathway has the potential to be targeted in the treatment of leukaemia with a high TRIB2 signature.

The impact of whey protein isolate on energy balance regulation

Using C57BL/6J mice fed whey protein isolate (WPI) enriched high fat (HFD) or low-fat diets (LFD), this study tested the hypothesis that WPI directly impacts on adiposity by influencing lipid metabolism. WPI suppressed HFD-induced body fat and increased lean mass at 8 weeks of dietary challenge despite elevated plasma triacylglycerol (TAG) levels, suggesting reduced TAG storage. WPI reduced HFD-associated hypothalamic leptin and insulin receptor (IR) mRNA expression, and prevented HFD-associated reductions in adipose tissue IR and glucose transporter 4 expression. These effects were largely absent at 21 weeks of HFD feeding, however WPI increased lean mass and cause a trend towards decreased fat mass, with notable increased Lactobacillus and decreased Clostridium gut bacterial species. Increasing the protein to carbohydrate ratio enhanced the above effects, and shifted the gut microbiota composition away from the HFD group. Seven weeks of WPI intake with a LFD decreased insulin signalling gene expression in the adipose tissue in association with an increased fat accumulation. WPI reduced intestinal weight and length, suggesting a potential functional relationship between WPI, gastro-intestinal morphology and insulin related signalling in the adipose. Extending the study to 15 weeks, did not affect adipose fat weight, but decreased energy intake, weight gain and intestinal length. The functionality of protein sensing lysophosphatidic acid receptor 5 (LPA5) in 3T3-L1 pre-adipocytes was assessed. Over-expression of the receptor in 3T3-L1 pre-adipocytes provided a growth advantage to the cells and suppressed cellular differentiation into mature fat cells. In conclusion, the data demonstrates WPI impacts on adiposity by influencing lipid metabolism in a temporal manner, resulting possibly due to changes in lean mass, hypothalamic and adipose gene expression, gut microbiota and gastrointestinal morphology. The data also showed LPA5 is a novel candidate in regulating of preadipocyte growth and differentiation, and may mediate dietary protein effects on adipose tissue.

Design history: exploring corporate communities

A design history is a narrative involving a multitude of social groups, interpretive flexibility, and eventual stabilization of shared understanding. Design history surfaces the practices that help shape and define engagements and can increase not only our theoretical understanding of what design is, but also our capacity to realize this understanding in practice. We use a design history perspective to examine how corporate technology initiatives establish and support open source communities and the crafting of relevant design practices that enable their advancement. We foster an evolving expression of design research that treats artifacts not as stable objects to be singularly evaluated, but as evolving systems contingent on historical trajectories.

The Vietnamese Corporate Bond Market An Early Exploration into the 1992-1999 Period

This paper summarizes the situation of corporate bonds in Vietnam for the period 1992-1999. Corporate bonds are new in the transitional economy, but the capital shortage and operational inefficiency of the banking sector and financial system would likely drive the bond market up in the future. The paper also discusses some conditions for the Vietnamese bond market to further develop, based on the facts and observation.

Mergers and Acquisitions in Vietnam’s Emerging Market Economy, 1990-2009

This paper is the first major and thorough study on the M&A activities in Vietnam’s emerging market economy, covering almost entirely the M&A history after the launch of Doi Moi. The surge in these activities since mid-2000s by no means incidentally coincides with the jump in FDI and FPI inflows into the nation. M&A industry in Vietnam has its socio-cultural traits that could help explain economic happenings, with anomalies and transitional characteristics, far better than even the most complete set of empirical data. Proceeds from sales of existing assets and firms have mainly flowed into the highly speculative industries of securities, banking, non-bank financials, portfolio investments and real estates. The impacts of M&A on Vietnam’s long-term prosperity are, thus, highly questionable. An observable high degree of volatility in the M&A processes would likely blow outthe high ex ante expectations by many speculators, when ex post realizations finally arrive. The effect of the past M&A evolution in Vietnam has been indecisively positive or negative, with significant presence of rent-seeking and likelihood of causing destructive entrepreneurship. From a socio-economic and cultural view, the degree of positive impacts it may result in for domestic entrepreneurship will perhaps be the single most important indicator.

Co-regulation of nuclear respiratory factor-1 by NFkappaB and CREB links LPS-induced inflammation to mitochondrial biogenesis.

The nuclear respiratory factor-1 (NRF1) gene is activated by lipopolysaccharide (LPS), which might reflect TLR4-mediated mitigation of cellular inflammatory damage via initiation of mitochondrial biogenesis. To test this hypothesis, we examined NRF1 promoter regulation by NFκB, and identified interspecies-conserved κB-responsive promoter and intronic elements in the NRF1 locus. In mice, activation of Nrf1 and its downstream target, Tfam, by Escherichia coli was contingent on NFκB, and in LPS-treated hepatocytes, NFκB served as an NRF1 enhancer element in conjunction with NFκB promoter binding. Unexpectedly, optimal NRF1 promoter activity after LPS also required binding by the energy-state-dependent transcription factor CREB. EMSA and ChIP assays confirmed p65 and CREB binding to the NRF1 promoter and p65 binding to intron 1. Functionality for both transcription factors was validated by gene-knockdown studies. LPS regulation of NRF1 led to mtDNA-encoded gene expression and expansion of mtDNA copy number. In cells expressing plasmid constructs containing the NRF-1 promoter and GFP, LPS-dependent reporter activity was abolished by cis-acting κB-element mutations, and nuclear accumulation of NFκB and CREB demonstrated dependence on mitochondrial H(2)O(2). These findings indicate that TLR4-dependent NFκB and CREB activation co-regulate the NRF1 promoter with NFκB intronic enhancement and redox-regulated nuclear translocation, leading to downstream target-gene expression, and identify NRF-1 as an early-phase component of the host antibacterial defenses.

Cytokinesis proteins Tum and Pav have a nuclear role in Wnt regulation.

Wg/Wnt signals specify cell fates in both invertebrate and vertebrate embryos and maintain stem-cell populations in many adult tissues. Deregulation of the Wnt pathway can transform cells to a proliferative fate, leading to cancer. We have discovered that two Drosophila proteins that are crucial for cytokinesis have a second, largely independent, role in restricting activity of the Wnt pathway. The fly homolog of RacGAP1, Tumbleweed (Tum)/RacGAP50C, and its binding partner, the kinesin-like protein Pavarotti (Pav), negatively regulate Wnt activity in fly embryos and in cultured mammalian cells. Unlike many known regulators of the Wnt pathway, these molecules do not affect stabilization of Arm/beta-catenin (betacat), the principal effector molecule in Wnt signal transduction. Rather, they appear to act downstream of betacat stabilization to control target-gene transcription. Both Tum and Pav accumulate in the nuclei of interphase cells, a location that is spatially distinct from their cleavage-furrow localization during cytokinesis. We show that this nuclear localization is essential for their role in Wnt regulation. Thus, we have identified two modulators of the Wnt pathway that have shared functions in cell division, which hints at a possible link between cytokinesis and Wnt activity during tumorigenesis.

Folate regulation of axonal regeneration in the rodent central nervous system through DNA methylation.

The folate pathway plays a crucial role in the regeneration and repair of the adult CNS after injury. Here, we have shown in rodents that such repair occurs at least in part through DNA methylation. In animals with combined spinal cord and sciatic nerve injury, folate-mediated CNS axon regeneration was found to depend on injury-related induction of the high-affinity folate receptor 1 (Folr1). The activity of folate was dependent on its activation by the enzyme dihydrofolate reductase (Dhfr) and a functional methylation cycle. The effect of folate on the regeneration of afferent spinal neurons was biphasic and dose dependent and correlated closely over its dose range with global and gene-specific DNA methylation and with expression of both the folate receptor Folr1 and the de novo DNA methyltransferases. These data implicate an epigenetic mechanism in CNS repair. Folic acid and possibly other nontoxic dietary methyl donors may therefore be useful in clinical interventions to promote brain and spinal cord healing. If indeed the benefit of folate is mediated by epigenetic mechanisms that promote endogenous axonal regeneration, this provides possible avenues for new pharmacologic approaches to treating CNS injuries.