ATP-sensitive potassium (KATP) channels have crucial roles in several biological processes. KATP channels possess four regulatory sulfonylurea receptors. The SUR proteins are members of the ubiquitous ATP-binding cassette (ABC) superfamily. However, unlike most ABC proteins, SURs do not transport substrates but function strictly as regulators of KATP channel activity. Currently, studies into the molecular basis by which various mutations in SUR2A cause disease are highly limited. This is primarily a consequence of poor solubility of isolated SUR2A NBDs, as is typical for many eukaryotic NBDs. By employing structure-based sequence alignments and biophysical studies, we determined domain boundaries for SUR2A NBD1 that enabled, for the first time, NMR studies of NBD1. Our biophysical studies demonstrate that the isolated SUR2A NBD1 is folded and exhibits differential dynamics upon ATP binding activity. Additional studies are now possible to examine the effects of disease-causing mutations on structure, dynamics, and interactions of NBD1.

SUR2A is a member of the ABC transporter superfamily. SUR2A mediated regulation of KATP channels is essential as mutations in the nucleotide binding domains (NBDs) of SUR2A are associated with cardiovascular disorders. Studies of eukaryotic NBDs, such as SUR2A, are hindered by low solubility of the isolated domain. We hypothesized that the solubility of heterologously expressed SUR2A NBDs depends on the definition of the domain boundaries. Boundaries were initially predicted using a combination of a structure-based sequence alignment and homology modeling, and subsequently verified by testing the solubility of five SUR2A NBD1 constructs with different N- or C-terminal boundaries. The boundaries of SUR2A NBD1 essential for solubility were identified. CD and NMR data indicate that SUR2A NBD1 is folded. Our method may be applied as a general method for developing suitable constructs of other NBDs of ABC proteins such as SUR isoforms, SUR2B and SUR2C, and the vacuolar transporter, Ycf1p.

ATP-sensitive potassium (KATP) channels have crucial roles in several biological processes. KATP channels possess four regulatory sulfonylurea receptors. The SUR proteins are members of the ubiquitous ATP-binding cassette (ABC) superfamily. However, unlike most ABC proteins, SURs do not transport substrates but function strictly as regulators of KATP channel activity. Currently, studies into the molecular basis by which various mutations in SUR2A cause disease are highly limited. This is primarily a consequence of poor solubility of isolated SUR2A NBDs, as is typical for many eukaryotic NBDs. By employing structure-based sequence alignments and biophysical studies, we determined domain boundaries for SUR2A NBD1 that enabled, for the first time, NMR studies of NBD1. Our biophysical studies demonstrate that the isolated SUR2A NBD1 is folded and exhibits differential dynamics upon ATP binding activity. Additional studies are now possible to examine the effects of disease-causing mutations on structure, dynamics, and interactions of NBD1.

The protein SiaA (Streptococcal iron acquisition) is involved in heme uptake in the bacterium Streptococcus pyogenes. It is difficult to obtain this protein in its fully holo form (completely loaded with heme). To increase the concentration of heme in the growing cell, we added ä-aminolevulinic acid (ALA) and ferrous sulfate (FeSO4), precursors of heme, to the growth media. Neither increasing the concentration of heme in vivo, nor growth at lower temperature for longer times, increased the production of holoprotein. The classical method of measuring the concentration of heme in a newly discovered heme protein is cumbersome. We have developed an improved method, which gives a solution that is more stable and has a cleaner spectrum. With further development, this new technique may replace the classical assay. Background information on S. pyogenes, SiaA, ABC transporters, heme biosynthesis, and the pyridine hemochrome assay are described.

Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types.

Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types.

ATP-binding cassette transporters ABCA1 and ABCG1 initiate reverse cholesterol transport generating HDL particles, whereas ABCG5/G8 promote biliary cholesterol secretion thereby facilitating the last step of reverse cholesterol transport. Mutations in the leptin axis result in obesity and dyslipidemia in ob/ob and db/db mice. These mice have defective HDL clearance, increased plasma cholesterol and decreased biliary cholesterol elimination. My studies demonstrate that ABCG5/G8 protein is low in these animals and can be restored with caloric restriction or leptin replacement. To directly test whether ABCG5/G8 alone is able to correct reverse cholesterol transport defect, liver specific ABCG5/G8 expression was achieved in db/db mice by administration of adenoviral ABCG5 and ABCG8. Restoration of biliary cholesterol is able partially to correct dyslipidemia in obese mice, but only in the presence of ezetimibe, an inhibitor of cholesterol absorption. ABCG5/G8 is the body’s primary defense against toxic effects of plant sterols. Plant sterols are used as cholesterol lowering food supplements. However, increased plasma plant sterol concentrations are associated with vascular lesions in dyslipidemic patients and animals. My in vitro studies demonstrate that individual plant sterol alter ABCA1 and ABCG1 abundance, cholesterol efflux and inflammatory cytokine secretion in macrophage foam cells at levels found in humans that consume plant sterol supplements.

Linfomas pertencem a um grupo de neoplasias em que há proliferação monoclonal de linfócitos malignos, sendo uma das neoplasias mais frequentemente diagnosticadas em cães. Podem ser classificados quanto à forma anatômica em multicêntrico, mediastinal, digestório e extranodal. Dentre os extranodais, os linfomas cutâneos são classificados histologicamente como epiteliotrópicos e não epiteliotrópicos e são predominantemente de imunfenótipo T, com raros casos do tipo B. A principal característica histopatológica do linfoma epiteliotrópico em cães é o tropismo das células neoplásicas pela epiderme, mucosa ou estruturas anexas, enquanto o linfoma não epiteliotrópico é caracterizado pela infiltração dérmica e subcutânea sem invasão das estruturas anexas. Os linfomas cutâneos caninos têm progressão rápida, são considerados bastante agressivos e com mau prognóstico, com baixa taxa de resposta à quimioterapia. Um dos fatores que podem contribuir para isso é a resistência das células a múltiplas drogas e entre esses mecanismos de resistência estão o efluxo de drogas do meio intracelular para o extracelular por meio dos transportadores da família ABC, como a glicoproteína-P, MRP (multiple resistance protein) e BCRP (breast câncer resistance protein) e da LRP (lung resistance protein), uma proteína vault responsável pelo transporte nucleocitoplasmático. O objetivo deste estudo foi caracterizar imunofenotipicamente os linfomas cutâneos, a proliferação celular por meio do marcador Ki67, a expressão das proteínas de resistência glicoproteína-P, MRP, BCRP e LRP e avaliar a relação dessas proteínas com a sobrevida dos animais. Foi realizado um estudo retrospectivo com 21 casos de cães linfomas cutâneos com diagnóstico histopatológico. A técnica de imunoistoquímica foi utilizada para determinar a imunofenotipagem dos linfomas pelos marcadores CD3 e CD20, a proliferação celular por Ki67 e a expressão de glicoproteína-P, MRP, BCRP e LRP. Dos 21 animais, 38% tiveram diagnóstico histopatológico de linfoma epiteliotrópico, 52% eram linfomas não epiteliotrópicos, 5% dos casos de linfoma não tiveram epiteliotropismo definido e 5% foram classificados como neoplasia de células redondas. O imunofenótipo predominante foi CD3+CD20- (76%), 15% dos casos eram CD3-CD20+ e 9% eram CD3+CD20+. A mediana de células marcadas para Ki67 foi de 31%. Com relação aos marcadores de resistência a múltiplas drogas, a mediana da marcação de glicoproteína-P foi de 40%, a de LRP foi de 65% enquanto para MRP e BCRP, 19% e 23%, respectivamente. Os linfomas cutâneos não epiteliotrópicos foram mais frequentes que os epiteliotrópicos e o imunfenótipo predominante foi o T. A ocorrência de linfócitos CD3-CD20+ e CD3+CD20+ indica a necessidade de mais estudos e um painel mais amplo de anticorpos para subtipagem desses linfomas. A glicoproteína-P teve maior expressão nos linfomas não epiteliotrópicos do que nos epiteliotrópicos e não houve correlação entre as proteínas de resistência e o tempo de sobrevida dos animais, sugerindo que, além da biologia da neoplasia, outros mecanismos de resistência a múltiplas drogas diferente dos estudados possam ter um papel relevante na baixa resposta do linfoma cutâneo à quimioterapia. / Lymphoma is a group of blood cell tumors that develop from monoclonal proliferation of malignant lymphocytes. Lymphoma is the most frequent neoplasia in dogs and can be anatomically classified in multicentric, mediastinal, digestive and extranodal. Cutaneous lymphomas an extranodal type of lymphoma are classified histologically in epitheliotropic and non-epitheliotropic and are predominantly of T-cell immunophenotype, and rare cases of B cell phenotype. The main histopathological characteristic of epitheliotropic lymphoma in dogs is the tropism of neoplastic cells by the epidermis, mucosa or adjacent structures, while non-epitheliotropic lymphoma is characterized by dermal and subcutaneous infiltration without invasion of adjacent structures. Canine cutaneous lymphomas have rapid progression, are considered very aggressive and have poor prognosis. These dogs, usually have a low rate of response to chemotherapy which can be associated to an antineoplastic resistance. Among mechanisms of resistance are efflux of drugs from intracellular to extracellular through ABC family transporters such as P-glycoprotein, MRP (multple resistance protein) and BCRP (breast cancer resistance protein) and LRP (lung resistance protein), a vault protein responsible for nucleocytoplasmic transport. The aim of this study was to characterize immunophenotypically cutaneous lymphomas, measure cell proliferation using the Ki67 marker, the expression of resistance proteins P-glycoprotein, MRP, BCRP and LRP and to evaluate the relationship of these proteins with the survival of the animals. A retrospective study was performed with 21 cases of dogs with cutaneous lymphoma with histopathological diagnosis. Immunohistochemical was used to immunophenotyping of lymphomas by CD3 and CD20 markers, Ki67 cell proliferation, and P-glycoprotein, MRP, BCRP and LRP expression. Of the 21 animals, 38% had histopathological diagnosis of epitheliotropic lymphoma, 52% were non-epitheliotropic lymphomas, 5% of lymphoma cases had no definition and 5% were classified as round cell neoplasia. The predominant immunophenotype was CD3+CD20- (76%), 15% of the cases were CD3-CD20 + and 9% were CD3 + CD20 +. The median of cells labeled for Ki67 was 31%. Regarding the markers of resistance to multiple drugs, the median of the P-glycoprotein label was 40%, which 65% of LRP while for MRP and BCRP, 19% and 23%, respectively. Non-epitheliotropic cutaneous lymphomas were more frequent than epitheliotropic lymphomas and the predominant immunophenotype was T. The occurrence of CD3-CD20+ and CD3+CD20+ lymphocytes indicates the need for further studies and a wider panel of antibodies for subtyping these lymphomas. P-glycoprotein had higher expression in non-epitheliotropic lymphomas than in epitheliotropic lymphomas and there was no correlation between resistance proteins and survival time of the animals, suggesting that in addition to the biology of neoplasia other mechanisms of resistance to multiple drugs different from those studied may play a relevant role in the low response of cutaneous lymphoma to chemotherapy.

Lignin is a key component of plant secondary cell walls, providing strength to the plant and allowing water transport. Lignin is a polymer of monolignols that are synthesized in the cell and transported into the cellulose rich cell wall. The primary goal of this thesis is to understand the mechanism(s) of monolignol deposition during xylogenesis. The currently accepted theory is that monolignols are exported by Golgi-mediated vesicle delivery to the secondary cell wall. When this theory was re-examined using cryofixed developing pine, quantitative autoradiography showed that monolignols did not accumulate in Golgi but were rapidly translocated from cytosol to cell wall. This suggests alternative mechanisms, such as membrane transporters, work in monolignol export. ATP binding cassette (ABC) transporters were chosen because they transport other secondary metabolites and some ABC transporter encoding genes are highly expressed in lignifying cells. Four candidate ABC transporters were selected in Arabidopsis (ABCB11, ABCB14, ABCB15 from the ABCB/MDR subfamily and ABCG33 from the ABCG/PDR subfamily) and shown to have overlapping, high vasculature expression patterns. Mutants with T-DNA insertions in single ABC transporter genes had no change in lignification of inflorescence stems. However, a reduced polar auxin transport phenotype was detected in mutants of ABCB11, ABCB14 and ABCB15. An additional approach was the use of inhibitors of ABC transporters. A new assay, which was developed to quantify lignification in primary xylem of Arabidopsis roots, demonstrated that ABC inhibitors did not change lignin deposition. Monolignols are exported and polymerized in the polysaccharide matrix of the cell wall, which includes hemicelluloses that may organize monolignols during polymerization. Since diverse lignified cell types are enriched in either G- or S-lignin, I hypothesized that this pattern could reflect different hemicellulose distributions, which was examined using antibody labeling of xylans or mannans in hybrid poplar xylem. While xylans were generally distributed in all secondary cell walls, mannans were enriched in fibers but not in the ray and vessel walls. In summary, during secondary cell wall deposition, monolignols are exported by unknown transporter(s) rather than Golgi vesicles. In developing poplar wood, the monolignols are deposited into diverse hemicellulose domains in different cell types. / Science, Faculty of / Botany, Department of / Graduate

Activation of the NOD-like receptor protein 3 (NLRP3) inflammasome complex causes the processing and release of mature IL-1β, with mitochondria playing key roles in its assembly. An orally active NLRP3 inflammasome inhibitor would be a significant advance in therapy for IL-1β-driven diseases. To overcome both, the variability among primary immune cells and the limitations of genetic manipulation of differentiated human or murine macrophages, we developed a simplified, reliable and relevant cell-based model for studying the NLRP3 inflammasome using the undifferentiated human myelomonocytic cell line THP1. We established that undifferentiated THP1 cells are fully competent for activation of the NLRP3 inflammasome and production of IL-1β, without differentiation into macrophages. CP-456,773 is a potent and selective inhibitor of the NLRP3 inflammasome, and it is an analogue of glyburide, a sulfonylurea receptor (SUR) inhibitor. Despite the extensive experimental use of CP-456,773, its molecular target remains unknown. Here we tested the hypothesis that mitochondrial ABCb7 or ABCb10 could be the pharmacologic targets of CP-456,773. We optimized a viral shRNA transduction method for genetic manipulations in THP1 cells and generated ABCb7 and ABCb10 knockdown (KD) THP1 cells. We demonstrate that NLRP3 inflammasome activation and CP-456,773 pharmacology are not altered in ABCb7- or ABCb10-deficient THP1 cells. For ABCb10, we confirmed these results using CRISPR/CAS9-mediated ABCb10 knockout (KO) THP1 sub-lines. In studies of mitochondrial fitness, we found that a previously observed reduction in oxygen consumption rate (OCR) following nigericin treatment was completely blocked in NLRP3 KO cells. Our data demonstrating that CP-456,773 rescues the NLRP3-dependent nigericin-induced decline in OCR and protects undifferentiated THP1 cells from nigericin-induced pyroptosis are consistent with the possibility that the NLRP3 protein itself may be the molecular target of CP-456,773. Moreover, we showed that ABCb10 KO THP1 cells exhibit increased rates of basal ATP production and glycolysis, suggesting an important role for ABCb10 in mitochondrial metabolism. Finally, RNA-Seq analysis of ABCb7 and ABCb10 KD in undifferentiated THP1 cells indicate new functions for these proteins, including cell communication and migration, apoptosis and cell adhesion. Overall, our findings demonstrate that undifferentiated THP1 cells are an ideal system in which to study the NLRP3 inflammasome.