Browsing by Author "Louw, Ann"
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- ItemA study of the mechanism of contraceptive action of naturally occurring and synthetic phenyl aziridines in rats(Stellenbosch : Stellenbosch University, 1998) Louw, Ann; Stellenbosch University. Faculty of . Dept. of .
- ItemAbrogation of glucocorticoid receptor dimerization correlates with dissociated glucocorticoid behavior of compound A(HighWire Press, 2010-03) Robertson, Steven; Allie-Reid, Fatima; Vanden Berghe, Wim; Visser, Koch; Binder, Anke; Africander, Donita; Vismer, Michael; De Bosscher, Karolien; Hapgood, Janet; Haegeman, Guy; Louw, Ann; A-7620-2012Compound A (CpdA), a dissociated glucocorticoid receptor modulator, decreases corticosteroid-binding globulin (CBG), adrenocorticotropic hormone (ACTH), and luteneinizing hormone levels in rats. Whether this is due to transcriptional regulation by CpdA is not known. Using promoter reporter assays we show that CpdA, like dexamethasone (Dex), directly transrepresses these genes. Results using a rat Cbg proximal-promoter reporter construct in BWTG3 and HepG2 cell lines support a glucocorticoid receptor (GR)-dependent transrepression mechanism for CpdA. However, CpdA, unlike Dex, does not result in transactivation via glucocorticoid-responsive elements within a promoter reporter construct even when GR is co-transfected. The inability of CpdA to result in transactivation via glucocorticoid- responsive elements is confirmed on the endogenous tyrosine aminotransferase gene, whereas transrepression ability is confirmed on the endogenous CBG gene. Consistent with a role for CpdA in modulating GR activity, whole cell binding assays revealed that CpdA binds reversibly to the GR, but with lower affinity than Dex, and influences association of [3H]Dex, but has no effect on dissociation. In addition, like Dex, CpdA causes nuclear translocation of the GR, albeit to a lesser degree. Several lines of evidence, including fluorescence resonance energy transfer, co-immunoprecipitation, and nuclear immunofluorescence studies of nuclear localization- deficient GR show that CpdA, unlike Dex, does not elicit ligand-induced GR dimerization. Comparison of the behavior of CpdA in the presence of wild type GR to that of Dex with a dimerization-deficient GR mutant (GRdim) strongly supports the conclusion that loss of dimerization is responsible for the dissociated behavior of CpdA.
- ItemChemoprevention of LA7-induced mammary tumor growth by SM6Met, a well-characterized cyclopia extract(Frontiers, 2018) Oyenihi, Omolola R.; Krygsman, Annadie; Verhoog, Nicolette; De Beer, Dalene; Saayman, Michael J.; Mouton, Thys M.; Louw, Ann; Wong, Vincent Kam WaiBreast cancer (BC) is the leading cause of cancer-related deaths in women. Chemoprevention of BC by using plant extracts is gaining attention. SM6Met, a wellcharacterized extract of Cyclopia subternata with reported selective estrogen receptor subtype activity, has shown tumor suppressive effects in a chemically induced BC model in rats, which is known to be estrogen responsive. However, there is no information on the estrogen sensitivity of the relatively new orthotopic model of LA7 cell-induced mammary tumors. In the present study, the potential chemopreventative and side-effect profile of SM6Met on LA7 cell-induced tumor growth was evaluated, as was the effects of 17b-estradiol and standard-of-care (SOC) endocrine therapies, such as tamoxifen (TAM), letrozole (LET), and fulvestrant (FUL). Tumor growth was observed in the tumorvehicle control group until day 10 post tumor induction, which declined afterward on days 12–14. SM6Met suppressed tumor growth to the same extent as TAM, while LET, but not FUL, also showed substantial anti-tumor effects. Short-term 17b-estradiol treatment reduced tumor volume on days prior to day 10, whereas tumor promoting effects were observed during long-term treatment, which was especially evident at later time points. Marked elevation in serum markers of liver injury, which was further supported by histological evaluation, was observed in the vehicle-treated tumor control, TAM, LET, and long-term 17b-estradiol treatment groups. Alterations in the lipid profiles were also observed in the 17b-estradiol treatment groups. In contrast, SM6Met did not augment the increase in serum levels of liver injury biomarkers caused by tumor induction and no effect was observed on lipid profiles. In summary, the results from the current study demonstrate the chemopreventative effect of SM6Met on mammary tumor growth, which was comparable to that of TAM, without eliciting the negative side-effects observed with this SOC endocrine therapy. Furthermore, the results of this study also showed some responsiveness of LA7-induced tumors to estrogen and SOC endocrine therapies. Thus, this model may be useful in evaluating potential endocrine therapies for hormone responsive BC. Keywords: chemoprevention, Cyclopia, mammary tumor, phytoestrogen, tamoxifen, letrozole, fulvestrant
- ItemCyclopia extracts act as ERα antagonists and ERβ agonists, in vitro and in vivo(Public Library of Science (PLOS), 2013-11) Visser, Koch; Mortimer, Morne; Louw, AnnHormone replacement therapy associated risks, and the concomitant reluctance of usage, has instigated the search for new generations of estrogen analogues that would maintain estrogen benefits without associated risks. Furthermore, if these analogues display chemo-preventative properties in breast and endometrial tissues it would be of great value. Both the selective estrogen receptor modulators as well as the selective estrogen receptor subtype modulators have been proposed as estrogen analogues with improved risk profiles. Phytoestrogen containing extracts of Cyclopia, an indigenous South African fynbos plant used to prepare Honeybush tea may serve as a source of new estrogen analogues. In this study three extracts, P104, SM6Met, and cup-of-tea, from two species of Cyclopia, C. genistoides and C. subternata, were evaluated for ER subtype specific agonism and antagonism both in transactivation and transrepression. For transactivation, the Cyclopia extracts displayed ERα antagonism and ERβ agonism when ER subtypes were expressed separately, however, when co-expressed only agonism was uniformly observed. In contrast, for transrepression, this uniform behavior was lost, with some extracts (P104) displaying uniform agonism, while others (SM6Met) displayed antagonism when subtypes were expressed separately and agonism when co-expressed. In addition, breast cancer cell proliferation assays indicate that extracts antagonize cell proliferation in the presence of estrogen at lower concentrations than that required for proliferation. Furthermore, lack of uterine growth and delayed vaginal opening in an immature rat uterotrophic model validates the ERα antagonism of extracts observed in vitro and supports the potential of the Cyclopia extracts as a source of estrogen analogues with a reduced risk profile.
- ItemDisease- and treatment-associated acquired glucocorticoid resistance(BioScientifica, 2018-10-11) Wilkinson, Legh; Verhoog, Nicolette J. D.; Louw, AnnENGLISH ABSTRACT: The development of resistance to glucocorticoids (GCs) in therapeutic regimens poses a major threat. Generally, GC resistance is congenital or acquired over time as a result of disease progression, prolonged GC treatment or, in some cases, both. Essentially, disruptions in the function and/or pool of the glucocorticoid receptor α (GRα) underlie this resistance. Many studies have detailed how alterations in GRα function lead to diminished GC sensitivity; however, the current review highlights the wealth of data concerning reductions in the GRα pool, mediated by disease-associated and treatment-associated effects, which contribute to a significant decrease in GC sensitivity. Additionally, the current understanding of the molecular mechanisms involved in driving reductions in the GRα pool is discussed. After highlighting the importance of maintaining the level of the GRα pool to combat GC resistance, we present current strategies and argue that future strategies to prevent GC resistance should involve biased ligands with a predisposition for reduced GR dimerization, a strategy originally proposed as the SEMOGRAM–SEDIGRAM concept to reduce the side-effect profile of GCs.
- ItemGlucocorticoid receptor concentration and the ability to dimerize influence nuclear translocation and distribution(Elsevier, 2013-02) Robertson, Steven; Hapgood, Janet P.; Louw, AnnGlucocorticoid receptor (GR) concentrations and the ability of the GR to dimerize are factors which influence sensitivity to glucocorticoids. Upon glucocorticoid binding, the GR is actively transported into the nucleus, a crucial step in determining GR function. We examined the effects of GR concentration and the ability to dimerize on GR nuclear import, export and nuclear distribution using both live cell microscopy of GFP-tagged GR and immunofluorescence of untagged GR, with both wild type GR (GRwt) and dimerization deficient GR (GRdim). We found that the observed rate of GR nuclear import increases significantly at higher GR concentrations, at saturating concentrations of dexamethasone (10¯ 6 M) using GFP-tagged GR, while with untagged GR it is only discernable at sub-saturating ligand concentrations (10 ¯10 -10 ¯ 9 M). Loss of dimerization results in a slower observed rate of nuclear import (2.5- to 3.3-fold decrease for GFP-GRdim) as well as a decreased extent of GR nuclear localization (18–27% decrease for untagged GRdim). These results were linked to an increased rate of GR export at low GR concentrations (1.4- to 1.6-fold increase for untagged GR) and where GR dimerization is abrogated (1.5- to 1.7-fold increase for GFP-GRdim). Furthermore, GR dimerization was shown to be required for the appearance of discrete GC-dependent GR nuclear foci, the loss of which may explain the increased rate of GR export for the GRdim. The reduction in the observed rate of nuclear import and increased rate of nuclear export displayed at low GR concentrations and by the GRdim could explain the lowered glucocorticoid response under these conditions.
- ItemGR dimerization and the impact of GR dimerization on GR protein stability and half-life(Frontiers Media, 2019-07-17) Louw, Ann; Libert, ClaudePharmacologically, glucocorticoids, which mediate their effects via the glucocorticoid receptor (GR), are a most effective therapy for inflammatory diseases despite the fact that chronic use causes side-effects and acquired GC resistance. The design of drugs with fewer side-effects and less potential for the development of resistance is therefore considered crucial for improved therapy. Dimerization of the GR is an integral step in glucocorticoid signaling and has been identified as a possible molecular site to target for drug development of anti-inflammatory drugs with an improved therapeutic index. Most of the current understanding regarding the role of GR dimerization in GC signaling derives for dimerization deficient mutants, although the role of ligands biased toward monomerization has also been described. Even though designing for loss of dimerization hasmostly been applied for reduction of side-effect profile, designing for loss of dimerization may also be a fruitful strategy for the development of GC drugs with less potential to develop GC resistance. GC-induced resistance affects up to 30% of users and is due to a reduction in the GR functional pool. Several molecular mechanisms of GC-mediated reductions in GR pool have been described, one of which is the autologous down-regulation of GR density by the ubiquitin-proteasome-system (UPS). Loss of GR dimerization prevents autologous down-regulation of the receptor through modulation of interactions with components of the UPS and post-translational modifications (PTMs), such as phosphorylation, which prime the GR for degradation. Rational design of conformationally biased ligands that select for a monomeric GR conformation, which increases GC sensitivity through improving GR protein stability and increasing half-life, may be a productive avenue to explore. However, potential drawbacks to this approach should be considered as well as the advantages and disadvantages in chronic vs. acute treatment regimes.
- ItemIlluminating the interrelated immune and endocrine adaptations after multiple exposures to short immobilization stress by in vivo blocking of IL-6(American Physiological Society, 2006-12) Smith, C.; Wilson, N. W.; Louw, Ann; Myburgh, K. H.ACUTE PSYCHOLOGICAL STRESS is known to activate the hypothalamic- pituitary-adrenal (HPA) axis, resulting in transiently increased release of inflammatory cytokines (28) and glucocorticoids (45). In contrast, multiple exposures to stress lead to adaptive responses in target tissues such as liver, skeletal muscle, and immune cells. These responses may be influenced by the severity of the stressor and the duration of the stress exposure. The endocrine and cytokine responses are known to be interrelated but are complex and still incompletely understood.
- ItemImpact of glucocorticoid receptor density on ligand- independent dimerization, cooperative ligand-binding and basal priming of transactivation : a cell culture model(Public Library of Science -- PLoS, 2013-05) Robertson, Steven; Rohwer, Johann M.; Hapgood, Janet P; Louw, AnnGlucocorticoid receptor (GR) levels vary between tissues and individuals and are altered by physiological and pharmacological effectors. However, the effects and implications of differences in GR concentration have not been fully elucidated. Using three statistically different GR concentrations in transiently transfected COS-1 cells, we demonstrate, using co-immunoprecipitation (CoIP) and fluorescent resonance energy transfer (FRET), that high levels of wild type GR (wtGR), but not of dimerization deficient GR (GRdim), display ligand-independent dimerization. Whole-cell saturation ligand-binding experiments furthermore establish that positive cooperative ligand-binding, with a concomitant increased ligand-binding affinity, is facilitated by ligand-independent dimerization at high concentrations of wtGR, but not GRdim. The down-stream consequences of ligand-independent dimerization at high concentrations of wtGR, but not GRdim, are shown to include basal priming of the system as witnessed by ligand-independent transactivation of both a GRE-containing promoterreporter and the endogenous glucocorticoid (GC)-responsive gene, GILZ, as well as ligand-independent loading of GR onto the GILZ promoter. Pursuant to the basal priming of the system, addition of ligand results in a significantly greater modulation of transactivation potency than would be expected solely from the increase in ligand-binding affinity. Thus ligand-independent dimerization of the GR at high concentrations primes the system, through ligand-independent DNA loading and transactivation, which together with positive cooperative ligand-binding increases the potency of GR agonists and shifts the bio-character of partial GR agonists. Clearly GR-levels are a major factor in determining the sensitivity to GCs and a critical factor regulating transcriptional programs.
- ItemInhibition of corticosteroid-binding globulin gene expression by glucocorticoids involves C/EBPβ(2014-10) Verhoog, Nicolette; Allie-Reid, Fatima; Vanden Berghe, Wim; Smith, Carine; Haegeman, Guy; Hapgood, Janet; Louw, AnnCorticosteroid-binding globulin (CBG), a negative acute phase protein produced primarily in the liver, is responsible for the transport of glucocorticoids (GCs). It also modulates the bioavailability of GCs, as only free or unbound steroids are biologically active. Fluctuations in CBG levels therefore can directly affect GC bioavailability. This study investigates the molecular mechanism whereby GCs inhibit the expression of CBG. GCs regulate gene expression via the glucocorticoid receptor (GR), which either directly binds to DNA or acts indirectly via tethering to other DNA-bound transcription factors. Although no GC-response elements (GRE) are present in the Cbg promoter, putative binding sites for C/EBPβ, able to tether to the GR, as well as HNF3α involved in GR signaling, are present. C/EBPβ, but not HNF3α, was identified as an important mediator of DEX-mediated inhibition of Cbg promoter activity by using specific deletion and mutant promoter reporter constructs of Cbg. Furthermore, knockdown of C/EBPβ protein expression reduced DEX-induced repression of CBG mRNA, confirming C/EBPβ’s involvement in GC-mediated CBG repression. Chromatin immunoprecipitation (ChIP) after DEX treatment indicated increased co-recruitment of C/EBPβ and GR to the Cbg promoter, while C/EBPβ knockdown prevented GR recruitment. Together, the results suggest that DEX repression of CBG involves tethering of the GR to C/EBPβ.
- ItemInhibition of cytochrome P450c11 by biogenic amines and an aziridine precursor, 2-(4-acetoxyphenyl_-2-chloro-n-methyl-ethylammonium chloride(Informa Healthcare, 2000) Louw, Ann; Allie, F.; Swart, A. C.; Swart, P.The interaction of several biogenic amines and Compound A (2-(4-acetoxyphenyl)-2-chloro- N-methyl-ethylammonium chloride), an analogue of the active substance in a HPLC fraction isolated from the shrub, Salsola tuberculatiformis Botsch., with cytochrome P450c11 was investigated. Noradrenaline, octopamine and Compound A inhibited the type I DOC induced difference spectrum of P450c11 and elicited a type II difference spectrum when added alone. The Ks-values for noradrenaline, octopamine, and Compound A were 0.8 mM, 0.16 mM and 0.36 mM, respectively. Dopamine, adrenaline and synephrine did not interact with, or inhibit, P450c11. Further investigation of Compound A indicated that it is a mixed inhibitor of sheep P450c11 with a stronger competitive (Kic = 106-110 µM) than uncompetitive (Kiu = 667-737 µM) element.
- ItemInterplay of the inflammatory and stress systems in a hepatic cell line : interactions between glucocorticoid receptor agonists and interleukin-6(HighWire Press, 2010-11) Visser, Koch; Smith, Carine; Louw, AnnThe liver plays an important role in inflammation and stress by producing the acute phase proteins (APPs) required for resolution of inflammation as well as by delivering systemic glucose, through gluconeogenesis, required to fuel the stress response. Disruption of the interplay between interleukin 6 (IL-6) and glucocorticoids (GCs), the peripheral mediators of inflammation and stress, respectively, maylead to side-effects associated with the pharmacological use of GCs.Thecurrent study investigated the interplay between IL-6 and GCs in a hepatoma cell line (BWTG3) at protein (protein activity assays, Western blotting, and ELISA) andmRNA(qPCR) levels. Specifically, the action of dexamethasone (Dex), a known antiinflammatory drug and glucocorticoid receptor (GR) agonist, is compared to that of Compound A (CpdA), a selective glucocorticoid receptor agonist (SEGRA). CpdA, like IL-6, but unlike Dex, increases GR binding and decreases the metabolic enzymes, tyrosine aminotransferase, phosphoenolpyruvate carboxykinase, andgammaglutamyltransferase, at protein ormRNAlevel. Like Dex, both CpdA and IL-6 increase the positive APPs, serum amyloid A and C-reactive protein, and decrease the negative APP, corticosteroid binding globulin. The study shows that the GC, Dex, and IL-6 generally have divergent effects on the GR and metabolic enzymes, while their functions are convergent on the APPs. In contrast to Dex,CpdAhas effects convergent to that of IL-6onthe GR, metabolic enzymes,and APPs. Thus these findings suggest that CpdA, like Dex, modulates APPs, leading to effective control of inflammation, while, in contrast to Dex, it is less likely to lead to GC-induced side-effects.
- ItemNot all progestins are the same : implications for usage(Elsevier, 2004-11) Hapgood, Janet P.; Koubovec, Dominique; Louw, Ann; Africander, DonitaRecent clinical evidence showing unexpected side-effects of progestins used in contraception and hormone replacement therapy has highlighted the importance of choice of synthetic progestin. The molecularmechanisms of action of the relatively nonspecific and most widely used synthetic progestins, medroxyprogesterone acetate and norethisterone, are discussed in the context of this recent clinical evidence. Future directions involving a more mechanism-based approach for improved therapeutics, with greater specificity and fewer side-effects, are discussed.
- ItemNovel role for receptor dimerization in post-translational processing and turnover of the GRα(Nature, 2018) Louw, Ann; Wilkinson, Legh; Verhoog, NicoletteGlucocorticoids (GCs), acting via the glucocorticoid receptor (GRα), remain the mainstay therapeutic choice for the treatment of inflammation. However, chronic GC use, aside from generating undesirable side-effects, results in GRα down-regulation, often coupled to a decrease in GC-responsiveness, which may culminate in acquired GC resistance. The current study presents evidence for a novel role of the dimerization state of the GRα in mediating GC-mediated GRα turnover. Through comparing the effects of dimerization promoting GCs on down-regulation of a transfected human wild type GRα (hGRwt) or a dimerization deficient GRα mutant (hGRdim), we established that a loss of receptor dimerization restricts GRα turnover, which was supported by the use of the dimerization abrogating Compound A (CpdA), in cells containing endogenous GRα. Moreover, we showed that the dimerization state of the GRα influenced the post-translational processing of the receptor, specifically hyper-phosphorylation at Ser404, which influenced the interaction of GRα with the E3 ligase, FBXW7α, thus hampering receptor turnover via the proteasome. Lastly, the restorative effects of CpdA on the GRα pool, in the presence of Dex, were demonstrated in a combinatorial treatment protocol. These results expand our understanding of factors that contribute to GC-resistance and may be exploited clinically.
- ItemPhytoestrogenic potential of Cyclopia extracts and polyphenols(Georg Thieme Verlag, 2013-05) Louw, Ann; Joubert, Elizabeth; Visser, KochCyclopia Vent. species, commonly known as honeybush, are endemic to Southern Africa. The plant is traditionally used as an herbal tea but several health benefits have recently been recorded. This minireview presents an overview of polyphenols found in Cyclopia and focusses on the phytoestrogenic potential of selected polyphenols and of extracts prepared from the plant.
- ItemSalsola tuberculatiformis botschantzev and an aziridine precursor analog mediate the in vivo increase in free corticosterone and decrease in corticosteroid-binding globulin in female wistar rats(The Endocrine Society, 1999-05) Louw, Ann; Swart, P.Salsola tuberculatiformis Botschantzev causes prolonged gestation in sheep and contraception in rats. An active fraction isolated from the shrub, containing a highly labile hydoxyphenyl aziridine or precursor, and a more stable analog, compound A, inhibits sheep adrenal cytochrome P450c11. In addition, compound A has been shown to bind to and be stabilized by corticosteroid-binding globulin (CBG). Binding may result in concomitant displacement of endogenous steroids, which could contribute to the biological effects of these compounds. The present study was undertaken to establish which mechanism would predominate in female rats. Compound A significantly (P,0.01) displaced glucocorticoids, but not progesterone, from corticosterone in both S. tuberculatiformis (P , 0.05)- and compound A (P , 0.01)-treated rats was also significantly higher due to displacement from CBG. In addition, both ACTH and CBG concentrations were significantly (P , 0.05) lower than control values. The levels of the gonadotropins were also reduced during treatment, but only LH values significantly (P , 0.05) so. These results suggest that binding of the test substances to CBG in female rat plasma and concomitant displacement of endogenous corticosterone could be part of the contraceptive mechanism of S. tuberculatiformis and the aziridine precursor, compound A.
- ItemScreening of four Cyclopia (honeybush) species for putative phyto-oestrogenic activity by oestrogen receptor binding assays(AOSIS OpenJournals, 2007) Verhoog, N. J. D.; Joubert, E.; Louw, AnnPhyto-oestrogens mediate an oestrogenic effect through binding to the oestrogen receptor (ER) subtypes, ERα and ERβ. Four commercially available Cyclopia (honeybush) species - C. intermedia, C. subternata, C. genistoides and C. sessiliflora - together with nine commercially obtainable polyphenols present in some or all of the species, were screened for phyto-oestrogenic activity, using a competitive whole-cell ER binding assay. Only naringenin, formononetin and luteolin were able significantly to displace 3H-E2 from hERα, whereas luteolin, naringenin, formononetin, eriodictyol, narirutin and eriocitrin displaced 3H-E2 from hERβ. Mangiferin, hesperidin and hesperetin did not bind to either receptor subtype. To our knowledge, this is the first time that binding of eriodictyol, eriocitrin and narirutin to the hERβ has been shown. Furthermore, both aqueous and methanol extracts from three independent harvestings of each Cyclopia species were screened. The results suggest that C. genistoides and C. subternata display significant phyto-oestrogenic activity and that methanol extracts from 'unfermented' (unoxidized) plant material generally display greater activity. Great variation exists within a species, however, with one C. genistoides harvesting displacing 3H-E2 from both ER subtypes, while another harvesting displaced 3H-E2 from only hERβ, and a third did not displace 3H-E2 from either receptor subtype.
- ItemSouth African herbal teas : Aspalathus linearis, Cyclopia spp. and Athrixia phylicoides - a review(Elsevier, 2008-10) Joubert, E.; Gelderblom, W. C. A.; Louw, Ann; De Beer, D.Rooibos (Aspalathus linearis (Brum.f) Dahlg.) and honeybush (Cyclopia Vent. species) are popular indigenous South African herbal teas enjoyed for their taste and aroma. Traditional medicinal uses of rooibos in South Africa include alleviation of infantile colic, allergies, asthma and dermatological problems, while a decoction of honeybushwas used as a restorative and as an expectorant in chronic catarrh and pulmonary tuberculosis. Traditional medicinal uses of Athrixia phylicoides DC., or bush tea, another indigenous South African plant with very limited localised use as herbal tea, include treatment of boils, acne, infected wounds and infected throats. Currently rooibos and honeybush are produced for the herbal tea market, while bush tea has potential for commercialisation. A summary of the historical and modern uses, botany, distribution, industry and chemical composition of these herbal teas is presented. A comprehensive discussion of in vitro, ex vivo and in vivo biological properties, required to expand their applications as nutraceutical and cosmeceutical products, is included, with the mainemphasis on rooibos. Future research needs include more comprehensive chemical characterisation of extracts, identification of marker compounds for extract standardisation and quality control, bioavailability and identification of bio-markers of dietary exposure, investigation of possible herb–drug interactions and plant improvement with regards to composition and bioactivity.
- ItemThe development of an anion exchanger for the isolation of human immunoglobulin for intravenous application(Stellenbosch : Stellenbosch University, 1984) Louw, Ann; Stellenbosch University. Faculty of . Dept. of .