PURPOSE: The purpose of this study was to compare the rate of grade 3-4 immune related adverse events (irAEs) in patients with advanced metastatic melanoma treated with the combined anti-CTLA-4 and anti-PD-immune-therapy at the Dana Farber Cancer Institute(DFCI), to that of the published rate of grade 3-4 irAEs among patients treated with the same combination of check-point therapy in the pivotal phase II and phase III trials that led to the FDA approval of the combination regimen. This study also measures the tumor response with the Ipi-Nivo combination therapy and overall-survival of patients in the study cohort at DFCI. METHODS/PROCEDURES: This is a retrospective cohort study conducted at DFCI during 2014 to 2016 among stage III/IV melanoma patients treated outside of the clinical trials with the Ipi-Nivo combination therapy. Chart review of the electronic medical record(EMR) was conducted to abstract the data for this study. irAEs were graded and classified as per the NCI-CTCAE v.4.0 guidelines. The comparison of the rate of grade 3 4 toxicity in the clinical settings at DFCI and the clinical trials was performed using a one sample proportion hypothesis test. For efficacy assessment of tumor response, RECIST1.1 criterion was used to ascertain the best clinical response. RESULTS: During an overall follow-up period of 600 days, 52 patients were treated on expanded access protocol (EAP) and commercial Ipi-Nivo combination therapy at DFCI. The rate of grade 3-4 immune mediated toxicity for this cohort of patients treated outside of clinical trials was 32.6%. The average rate of grade 3-4 irAEs reported in phase II/III clinical trials was approximately 55%. The results from the one-proportion hypothesis test [(P-value: 0.002) (95% C.I: 19.14-46.23)], prove that patients in the “real world” clinical settings have a different safety profile than patients treated in the clinical trials. The rate of grade 3-4 irAEs was found to be lower (19.14% to 46.23%) in the population treated with Ipi/Nivo combination therapy at the DFCI, compared to the check-mate clinical trials (approximately 55%) CONCLUSION: The results from the study indicate a lower rate of grade 3-4 irAEs in patients treated at DFCI, in comparison with the patients treated in the clinical trials for the Ipi-Nivo combination group. The results support the need for preemptive safety signal detection of symptoms of irAEs to improve patient’s safety. However, larger database studies are required for the generalizability of this results to a wider patient population treated outside of DFCI.

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. Elevated intraocular pressure (IOP), resulting from increased aqueous humor outflow resistance, is a major risk factor for the development and progression of POAG. Outflow resistance in the trabecular outflow pathway is mainly (50-75%) generated in the juxtacanalicular connective tissue (JCT), and partially (25-50%) in the portion distal to the inner wall of Schlemm’s canal. The details of how aqueous humor flows through these tissues and how resistance in these tissues is regulated are not fully understood in normal and POAG eyes. Aqueous humor outflow was shown to be “segmental”, with discontinuous active regions of aqueous humor filtration along the trabecular outflow pathway that can be labeled with perfused fluorescent tracers and measured as effective filtration area (EFA). In this study, we investigated the relationship between changes in EFA along the trabecular outflow pathway and outflow facility/IOP under two experimental conditions. The first experiment was designed to increase outflow facility by using netarsudil, a recently approved Rho kinase inhibitor class glaucoma medication, in normal human donor eyes. The second experiment was designed to increase IOP with topical steroid treatment for 5 weeks in mice. The purpose of this study is to verify whether EFA can be modulated by netarsudil or steroid treatment and to demonstrate the morphological changes that may be responsible for the changes of EFA. We analyzed EFA along the trabecular outflow pathway and found that elevated/reduced EFA correlated with increased outflow facility/IOP. Guided by EFA, we performed detailed morphological comparison between the active and inactive portions of aqueous humor filtration tissue to evaluate possible structural changes involved in EFA regulation. We found that increased EFA was associated with a loosened JCT structure and dilated episclearal veins, while decreased EFA was associated with a compacted JCT structure, increased deposition of curly collagen and/or fibrillary structure in the trabecular meshwork, and increased basement membrane continuity. Our data suggest that the netarsudil/steroid-induced morphological changes in the trabecular outflow pathway can result in either an increase or decrease in EFA, which in turn contributes to the regulation of outflow facility/IOP. / 2020-10-24T00:00:00Z

Ziel der vorliegenden Arbeit war es, den Einfluss von unterschiedlich behandelten Sojabohnen in einer Broilerration (Mais und behandeltes Soja) auf die ernährungsphysiologischen Parameter beim wachsenden Küken zu prüfen. Unter Berücksichtigung der Einflussgrößen (z. B. Zeit, mechanischer Energieeinsatz und Futterstruktur), die die Effektivität der Futterbearbeitungsverfahren beeinflussen, wurde zunächst die Behandlung einer einheitlichen Charge von Vollfettsojabohnen unter folgenden technische Bedingungen vorgenommen: A: Walzenstuhl (WS) + Konditionierung (40 min./100 °C, Standardverfahren); B: WS + Konditionierung (40 min./100 °C) + Flockierung; C: WS + Konditionierung (10 min./100 °C); D: WS + Konditionierung (10 min. /100 °C) + Expandieren (20 kWh/t); E: Hammermühle (HM) + Konditionierung (10 min./100 °C) + Extrusion (15 kWh/t); F: HM + Konditionierung (10 min./100 °C), G: HM + Konditionierung (10 min./100 °C) + Expandieren (20 kWh/t); H: HM + Expandieren (20 kWh/t); I: HM + Expandieren (20 kWh/t) mit Dampfzufuhr; K: HM + Expandieren (40 kWh/t). Mit 6 ´ 10 männlichen Broilern der Herkunft Cobb wurde im Zeitraum vom 7. - 28. Lebenstag (LT) je Behandlung ein Wachstumsversuch durchgeführt. Während des Wachstumsversuchs wurden Daten zum Futterverzehr, Lebendmassezuwachs und Futteraufwand sowie Energie- und Proteinansatz über Körperanalyse erfaßt. Die ileale Aminosäureverdaulichkeit der einzelnen Mischungen wurde in jeweils 4 gepoolten Chymusproben (9 Tiere / gepoolte Probe) mit Hilfe eines Indikators (HCl-unlösliche Rohasche, Zusatz von 1 % Celite) nach Verfütterung vom 21. - 28. LT bestimmt. Parallel zum Wachstumsversuch wurde ein Stoffwechselversuch (6 männliche Küken der Herkunft Cobb je Behandlungsstufe) im Zeitraum vom 15. - 21. LT bei Fütterung der Versuchsmischung durchgeführt. Dabei erfolgte 3-mal täglich eine quantitative Exkrementsammlung. Neben der N- Bestimmung in den Exkrementen der Basis für die N-Bilanzmessung wurde der Bruttoenergiegehalt erfaßt und auf dieser Grundlage der Gehalt an N-korrigierter umsetzbarer Energie (MEn) in den Futtermischungen bestimmt. Weiterhin wurde aus den Daten der Bilanzmessungen der physiologische Nutzwert (PNu) als Kriterium der Proteinqualitätsbeurteilung sowie die Lysinwirksamkeit abgeleitet. Am Ende des Stoffwechselversuchs wurden die Tiere geschlachtet und die Trypsinaktivität im Chymus des Jejunums bestimmt. Die Ergebnisse lassen sich folgendermaßen zusammenfassen: 1. In Abhängigkeit von den gegebenen Bedingungen der Futterbehandlungsverfahren konnten unterschiedliche Trypsininhibitoraktivitäten (TIA) in den behandelten Sojabohnen erzielt werden. Eine starke Reduzierung des TIA war insbesondere bei den behandelten Sojabohnen A bis G erkennbar. Die relative Restaktivität lag bei diesen Gruppen (im Vergleich zur rohen Sojabohne) bei 16-27 %. Im Gegensatz dazu wurde eine höhere relative Restaktivität in den behandelten Sojabohnen H, I und K in Höhe von 75 %, 32 % und 48 % festgestellt . 2. Die Futteraufnahme, Lebendmassezunahme und Futterverwertung waren bei der Gruppe H infolge der höchsten Restaktivität des Trypsininhibitors signifikant niedriger als bei der Kontrolle A und anderen Gruppen (B, C, D, E, F, G, I und K). Analog dazu führte die höchste TIA in den Sojabohnen H ebenfalls zu einem signifikant verminderten Nährstoffansatz im Ganzkörper. Der Einfluss der höchsten Restaktivität des Trypsininhibitors auf den PNu war bei Gruppe H wenig ausgeprägt. 3. Die N-korrigierte umsetzbare Energie in den Futtermischungen B (14,87 MJ /kg T) und F (14,70 MJ /kg T) war signifikant höher als bei der Kontrolle A (13,96 MJ /kg). Der Grund für die Steigerung der N-korrigierten umsetzbaren Energie bei B und F kann dabei in der möglichen Verbesserung der Energieverfügbarkeit durch die Behandlungsverfahren Flockierung und Expander liegen. 4. Aufgrund der höchsten Restaktivität des Trypsininhibitors wurde bei der Gruppe H eine verminderte ileale Aminosäure- und Stickstoffverdaulichkeit im Vergleich zu Kontrolle A und den anderen Gruppen (B, C, D un E) festgestellt. Durch zusätzliche Dampfzufuhr und Erhöhung des Energieeintrages in den Gruppen I bzw. K konnten die antinutritiven Faktoren in den Sojabohnen reduziert werden und demzufolge die ileale Aminosäure- und Stickstoffverdaulichkeit bei den Gruppen I und K gegenüber der Gruppe H signifikant verbessert werden. Obwohl bei der Gruppe H die ileale Lysinverdaulichkeit gegenüber der Kontrolle A signifikant niedriger lag, wurde in Bezug auf die Lysinwirksamkeit kein signifikanter Unterschied zwischen den Gruppen H und der Kontrolle A festgestellt. 5. Die Trypsinaktivität im Chymus wurde auch in Abhängigkeit von der Restaktivität des Trypsininhibitors in den Sojabohnen unterschiedlich beeinflusst. Eine signifikant verminderte Trypsinaktivität im Chymus war insbesondere bei den Gruppen H sowie K feststellbar.

Magister Scientiae - MSc / Various dermatological disorders, such as formation of black pigmented patches on the surface of the skin arise from the over-activity of tyrosinase enzyme's degenerative action. This enzyme is further implicated in the involvement of melanin in malignant melanoma, the most lifethreatening skin tumors. Although, synthetic products were found effective to combat this menace, nevertheless, overtime detrimental effect on human skin is a challenge. Investigation of natural tyrosinase inhibitors from methanol extracts of medicinal plants of Lamiaceae family using L-tyrosine as substrate on three different complementary assays (TLC bio-autography, spectrophotometry and cyclic voltammetry) was carried out accordingly. The result indicated Salvia chamelaeagnea, Salvia dolomitica, Plectranthus ecklonii, Plectranthus namaensis, and Plectranthus zuluensis, with significant zone of inhibition against tyrosinase on TLC bio-autography, spectrophotometry result showed that extracts of Plectranthus ecklonii (IC50 = 21.58 μg/mL), Plectranthus zuluensis (IC50 = 23.99 μg/mL), Plectranthus madagascariensis (IC50 = 23.99 μg/mL) and Salvia lanceolata (IC50 = 28.83) demonstrated good anti-tyrosinase activity when compared with kojic acid (IC50 = 3.607 μg/mL). On the other hand, cyclic voltammetry are in consonant with above results thereby supported the nomination of some of the extracts as strong anti-tyrosinase agents. Salvia chamelaeagnea showed strong activity in cyclic voltammetry and clear zone of inhibition on TLC bioautography, these reasons gave us justification for further chemical study to isolate the bioactive constituents. Phytochemical investigation of the bioactive extract of Salvia chamelaeagnea using different chromatographic methods including column chromatographic and semi preparative HPLC afforded six (6) known compounds viz carsonol (C1), carnosic acid (C2), 7- ethoxylrosmanol (C3), ursolic acid (C4), rosmanol (C5) and ladanein (C6). Their chemical structures were elucidated by analyses of spectroscopic (1H and 13C NMR) data as well as correlations with existing literature. The methanolic extract of S. chamelaeagnea (SC) showed moderate antityrosinase (IC50 = 267.4 μg/mL) activity, total antioxidant capacities measured as: Oxygen radicals absorbance capacity (ORAC; 14970 ± 5.16 μM TE/g), ferric-ion reducing antioxidant power (FRAP; 9869.43 ± 7.87 μM AAE/g) and trolox equivalent absorbance capacity (TEAC; 13706.5 ± 0.95 μM TE/g). Excellent total antioxidant capacities were demonstrated by C1 and C5 respectively as FRAP (9338.92 ± 1.72; 8622.73 ± 1.92) μM AAE/g; TEAC (16505 ± 0.86; 10641.5 ± 0.52) μM TE/g; ORAC (14550.5 ± 3.65; 14633.90 ± 3.84) μM TE/g and including the inhibition of Fe2+ -induced lipid peroxidation (IC50 = 32.5; 30.25) μg/mL. All the compounds except C4 are electro-active with well-defined oxidation-reduction peaks while C1 demonstrated the highest tyrosinase inhibitory activity by strongly decreased the inhibition current with time using cyclic voltammetry method. The isolated compounds especially C1, C2 and C5 are well known to combat with ageing problems and documented for their powerful activity against oxidative stress and alzheimer's diseases, which are ageing related symptoms. The isolation of such bioactive compounds indicated the synergetic effect of the results of the three methods used in this thesis. This is the first report on the evaluation of both anti-tyrosinase and total antioxidant capacities of the isolated compounds from S. chamelaeagnea. The findings therefore can be used as background information for exploitation of skin depigmentation and antioxidant agents from natural source.

This thesis examined the effects of applied stabilized nitrogenous fertilizers with sulphur (ENSIN and ENTEC 26) and nitrogenous fertilizers without inhibitors (DASA) in the nutrition of winter wheat. The effect of various forms of stabilized nitrogenous fertilizers on the yield and qualitative parameters of winter wheat was studied during a three-year vegetative experiment. The experiment was carried out in the form of a small plot experiment in two localities (Žabčice near Brna and Vatín near Žďáru nad Sázavou). The experiment included the following six variants of fertilisation: 1. unfertilised control, 2. DASA + DASA (ammonium nitrate and ammonium sulphate) , 3. ENSIN, 4. ENTEC 26, 5. LAD (ammonium nitrate with dolomite) + ENSIN, 6. LAD + ENTEC 26. On all fertilized variants there was a significant increase in yeald compared to the unfertilized control (an average of 22.4 to 37.3 %). The highest average yield for three years amounted variant LAD + ENSIN in the locality Vatín and Žabčice. The maximum average yield 7,39 t/ha reached the option LAD + ENSIN in the locality Vatín and 7,61 t/ha in the locality Žabčice. This variant also achieved the highest content of N - substances and sedimentation value. Only in bulk density was better option LAD + ENTEC 26, however, there was no significant difference. Variant LAD + ENSIN was effective also in terms of economic efficiency.

A semente da Araucaria angustifolia, denominada pinhão, é consumida no Sul e Sudeste do Brasil, como farinha em pratos regionais ou cozida. Há relativamente pouca informação a respeito da composição química e do valor nutricional da semente e de sua farinha. Neste trabalho, a farinha de pinhão, obtida através de diferentes tratamentos térmicos foi avaliada como complemento protéico em um experimento biológico com ratos recém-desmamados. Os animais experimentais consumiram cinco dietas (n=6) com diferentes fontes de proteína: dieta com caseína (CAS), dieta com 80% caseína e 20% (w/w) proteína de farinha de pinhão (PF) sem tratamento térmico (NATPIN), considerando 33.1 ± 1.4 g de proteína/kg desta farinha, dieta com 80% caseína e 20% PF seca a 50°C por 16 horas (PF50), considerando 49.2 ± 0.6 g de proteína/Kg desta farinha, dieta com 80% caseína e 20% PF seca a 80°C por 16 horas (PF80), considerando 50.5 ± 0.8 g de proteína/kg desta farinha e dieta aprotéica (APROT). Os valores de ganho de peso, ingesta de alimento, Coeficiente de Eficácia Alimentar (PER) e Razão Protéica Líquida (NPR) foram similares para as dietas CAS e FP80. O escore químico de aminoácidos corrigido pela digestibilidade protéica (PDCAAS) da PF80 foi o mais alto nas farinhas de pinhão testadas. O escore químico (CS) da farinha de pinhão se mostrou semelhante ao encontrado em outros cereais, sendo a lisina o principal aminoácido limitante, seguida da histidina. Valores mais baixos em todos os parâmetros nutricionais foram encontrados nos animais alimentados com dietas onde foi usada a farinha de pinhão. A farinha de pinhão seca a 80°C por 16 horas mostrou resultados similares nos parâmetros nutricionais ao grupo CAS, e pode ser utilizada, substituindo até 20% de uma proteína de alto valor biológico (AVB) em formulações alimentares. / The seeds of Araucaria angustifolia, namely “pinhão”, are consumed in South and Southeast of Brazil, like flour in regional dishes or baked. There is relatively few information about the chemical composition and nutritional value of the seed and its flour. In this work, “pinhão” flour obtained by different heat treatments was evaluated as an additive in biological experiment for growing rats. Wistar rats were fed five experimental diets (n=6) containing different protein sources: only casein (CAS), diet with 80% casein supplemented with 20% (w/w) “pinhão” flour (PF) protein without heat treatment (NATPIN), considering 33.1 ± 1.4 g of protein/kg of this flour, diet with 80% casein and 20% PF dried at 50°C for 16 hours (PF 50), considering 49.2 ± 0.6 g of protein/kg of this flour and diet with 80% casein and diet with 20% PF dried at 80°C for 16 hours (PF80), considering 50.5 ± 0.8 g of protein/kg of this flour and one aproteic diet (APROT). Values for weight gain, feed ingest, Protein Efficiency Ratio (PER) and Net Protein Ratio (NPR) were similar for diets CAS and PF80, and acoording to this, the Protein Digestibility Corrected Amino acid Score (PDCAAS) of PF80 was the highest of all the “pinhão” flours tested. The Chemical Score (CS) of “pinhão” flour showed similarity to the results found for other cereals, being lysine the limiting amino acid, folowed by histidine. Lowest values for all nutritional parameters were observed for diets complemented with “pinhão” flour. “Pinhão” flour heated at 80°C for 16 hours and used as supplementary in diet had the most similar results in all nutritional parameters to casein-based diets, and can be used as complementary source, substituting until 20% of a high biological value protein in food formulations.

DIAS, Lucas Pinheiro. Purificação e caracterização de um inibidor de tripsina das flores de Cassia fistula Linn. com atividade antimicrobiana. 2012. 83 f. Dissertação (Mestrado em Bioquímica)-Universidade Federal do Ceará, Fortaleza-CE, 2012. / Submitted by Eric Santiago (erichhcl@gmail.com) on 2016-07-19T13:34:23Z No. of bitstreams: 1 2012_dis_lpdias.pdf: 2004862 bytes, checksum: cd42d65281498494806d7ca987015da7 (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T20:29:09Z (GMT) No. of bitstreams: 1 2012_dis_lpdias.pdf: 2004862 bytes, checksum: cd42d65281498494806d7ca987015da7 (MD5) / Made available in DSpace on 2016-08-02T20:29:09Z (GMT). No. of bitstreams: 1 2012_dis_lpdias.pdf: 2004862 bytes, checksum: cd42d65281498494806d7ca987015da7 (MD5) Previous issue date: 2012 / Plants synthesize proteins that have antimicrobial properties, which can be used to substitute chemical pesticides in agriculture and as new drugs for the control of bacterial infections in humans. Among the various plant structures, the flowers seem to be a promising source of active molecules against pathogens, particularly if considered its important physiological role, which should be preserved. Therefore, this experimental research aimed at the prospection of novel proteins with antimicrobial activity in wild flowers and to subsequent purification, biochemical characterization and evaluation of antimicrobial activity of a trypsin inhibitor present in flowers of Cassia fistula Linn (the golden shower tree). The total extract of C. fistula flowers was prepared in 50 mM sodium phosphate buffer, pH 7.5. This extract presented trypsin inhibitory activity (42.41 ± 0.35 IU/mgP) and papain (27.10 ± 0.23 IU/mgP), besides to the presence of peroxidase (20.0 ± 0.18 UAP/mgP) and chitinase (1.70 ± 0.21 ηkatal/mgP). On the other hand, the hemagglutinating, β-1,3-glucanase, protease and urease activities were not detected. The trypsin inhibitor of C. fistula, named CfTI, was purified by fractionating the crude extract with trichloroacetic acid (2.5%) followed by affinity (anidrotripsina-Sepharose-4B) and reverse phase (Vydac C-18TP 522) chromatographies. CfTI is a glycoprotein with an apparent molecular mass of 22.2 kDa, pI 5.0 and NH2-terminal sequence showing high similarity with Kunitz soybean trypsin inhibitor (SBTI). The inhibitor was not stable to heat, and loss 23.4% when incubated at 60 °C for 15 minutes. However, it proved to be stable to changes of pH. CfTI (100 µg/mL) slowed the growth of pathogenic fungi of agricultural importance, Colletotrichum lindemuthianum and Fusarium solani, and also presented antibacterial activity against the human pathogenic bacteria, Staphylococcus aureus and Enterobacter aerogenes. The results demonstrate the potential of the flowers as a source of diverse bioactive proteins, as the trypsin inhibitor present in C. fistula flowers, promoting its biotechnological potential application against fungi and bacteria of relevance to Agriculture and human health. / As plantas sintetizam proteínas que possuem propriedades antimicrobianas, podendo ser utilizadas em substituição aos defensivos químicos no campo e como fonte de novas drogas para o controle de infecções bacterianas em humanos. Dentre as diversas estruturas vegetais, órgãos reprodutivos como as flores parecem ser uma fonte promissora de tais moléculas ativas contra patógenos, particularmente se considerado o seu relevante papel fisiológico, o qual deve ser preservado. Nesse contexto, a presente pesquisa experimental teve como objetivos a prospecção de proteínas com ação antimicrobiana em flores silvestres e posterior purificação, caracterização bioquímica e avaliação da atividade antimicrobiana de um inibidor de tripsina presente em flores de Cassia fistula Linn. (Chuva-de-ouro). O extrato total das flores de C. fistula foi preparado em tampão fosfato de sódio 50 mM, pH 7,5. Esse extrato apresentou atividade inibitória de tripsina (42,41 ± 0,35 UI/mgP) e de papaína (27,10 ± 0,23 UI/mgP), além de mostrar a presença de peroxidase (20,0 ± 0,18 UAP/mgP) e quitinase (1,70 ± 0,21 ηkatal/mgP), estando ausentes as atividades hemaglutinante, β-1,3-glucanásica, ureásica e proteásica. O inibidor de tripsina de C. fistula, denominado CfTI, foi purificado do extrato total por fracionamento com ácido tricloroacético (2,5%), seguido de cromatografias de afinidade (anidrotripsina-Sepharose-4B) e fase reversa (Vydac C-18TP 522). CfTI é uma glicoproteína com massa molecular aparente de 22,2 kDa, pI 5,0 e sequência NH2-terminal exibindo alta similaridade com o inibidor de tripsina da soja do tipo Kunitz (SBTI). O inibidor mostrou-se pouco estável ao calor, reduzindo sua atividade inibitória para 23,4% quando incubado a 60 °C, durante 15 minutos. No entanto, ele se mostrou bastante estável a variações no pH. CfTI (100 µg/mL) retardou o crescimento dos fungos fitopatogênicos de importância agrícola, Colletotrichum lindemuthianum e Fusarium solani, além de apresentar atividade antibacteriana frente às bactérias patogênicas ao homem, Staphylococcus aureus e Enterobacter aerogenes. Os resultados obtidos demonstram o potencial das flores como fonte diversificada de proteínas bioativas, evidenciando o inibidor de tripsina presente em flores de C. fistula, fomentando sua aplicação biotecnológica frente a fungos e bactérias de relevância para a Agricultura e Saúde.

Introdução: A artrite reumatoide é uma doença inflamatória autoimune associada à complicações sistêmicas como fadiga e perda muscular. Perda muscular pode estar relacionada com a ativação do sistema ubiquitina-proteossomo. O objetivo deste trabalho foi avaliar a perda muscular e o evolvimento do proteossomo no modelo de artrite induzida por colágeno (CIA), com ou sem o tratamento de metotrexato ou inibidor de TNF (etanercepte). Métodos: Camundongos DBA1/J machos foram divididos em 4 grupos (n=8 cada): CIA (salina); ETN (etanercepte, 5.5 /) e MTX (metotrexato, 35 /), tratados duas vezes por semana por 6 semanas, e um grupo controle saudável (CO). Tratamentos iniciaram uma semana após a injeção do booster. Escore clínico, edema da pata traseira e peso corporal foram analisados durante o período experimental. Músculo gastrocnêmio (GA) foi pesado após a morte e usado para quantificar a atividade, níveis proteicos e expressão de mRNA das diferentes subunidades do proteossomo através de ensaio fluorogênico, Western blot e rtPCR, respectivamente. Resultados: Tratamentos reduziram o desenvolvimento da doença, observado através do menor escore clínico e edema da pata traseira nos grupos ETN e MTX. ETN apresentou maior peso corporal do que MTX nas semanas 5 e 7. Músculo GA estava aumentado em ETN do que CIA e MTX, um resultado também observado no peso muscular normalizado. As propriedades catalíticas do proteossomo 26S muscular mostraram um aumento na atividade do tipo caspase nos grupos CIA e MTX. Tecidos musculares de animais MTX demonstraram maiores níveis proteicos das subunidades do proteossomo PSMB8 e PSMB9 e maior expressão gênica de Psmb5, Psmb8 e Psmb9. Por outro lado, a expressão de Psmb6 estava diminuída e de Psmb9 estava aumentada em CIA. Conclusões: Apesar de ambos os medicamentos melhorarem o escore da doença, ETN apresentou um afeito anti-artrítico mais forte e foi o único tratamento capaz de prevenir parcialmente a perda muscular. Ao contrário de ETN, CIA e o tratamento com MTX apresentaram perda muscular e atividade e expressão do proteossomo aumentadas. / Background: Rheumatoid arthritis is an autoimmune inflammatory disease associated with systemic complications like fatigue and muscle wasting. Muscle wasting could be related to the activation of the ubiquitin-proteasome system. The aim of this study was to evaluate muscle loss and involvement of the proteasome in collagen-induced arthritis (CIA), with or without treatment with methotrexate or a TNF inhibitor (etanercept). Methods: Male DBA1/J mice were divided into 4 groups (n=8 each): CIA (saline); ETN (etanercept, 5.5 /) and MTX (methotrexate, 35 /), treated twice a week for 6 weeks, and a healthy control group (CO). Treatments started one week after booster injection. Clinical score, hind paw oedema, and body weight were analysed during the experimental period. Gastrocnemius muscles (GA) were weighted after death and used to quantify proteasome activity, protein levels and mRNA expression of its subunits by Western blot and rtPCR, respectively. Results: Treatments slowed disease development, observed through smaller clinical score and hindpaw edema in ETN and MTX groups. ETN presented higher body weight compared to MTX group at weeks 5 and 7. GA weight was heavier in ETN than CIA and MTX, a result also observed in the normalized muscle weight. The catalytic properties of 26S proteasome showed an increase of caspase-like activity in CIA and MTX groups. Muscles tissues of MTX treated animals showed higher protein levels for proteasomal subunits PSMB8 and PSMB9 and higher gene expression for Psmb5, Psmb8 and Psmb9. In contrast, expression of Psmb6 was decreased and of Psmb9 was enhanced in CIA. Conclusions: Although both drugs improved the disease score, ETN presented a stronger anti-arthritic effect and was the only treatment able to partially prevent muscle wasting. In contrast to ETN, CIA and MTX treatment did not prevent muscles loss due to increased proteasome expression and activity.

F-box only protein 7 (Fbxo7), a member of the F-box-only subfamily of FBPs, is a biologically and pathophysiologically important human protein that assumes many critical functions. The different functions of Fbxo7 depend on the formation of various multi-protein complexes. Possible interplay between different Fbxo7 functions further complicate the protein-protein interaction networks involved in Fbxo7 biology. Although significant progresses have been made to understand the functions, regulation, specificity, and protein interaction network of Fbxo7, a myriad of questions remain to be answered. The objectives of the work presented in this dissertation are to elucidate the molecular structures underlying the functions of Fbxo7 and the interaction with its protein partners, such as proteasome inhibitor PI31. The best known biological function of Fbxo7 is its role as the substrate-recognition subunit of the SCFFbxo7 (Skp1-Cul1-F-box protein) E3 ubiquitin ligase that catalyzes the ubiquitination of hepatoma up-regulated protein (HURP) and inhibitor of apoptosis protein (IAP). Fbxo7 also assumes various SCF-independent functions through interact with its protein partners that are not the substrates of the ubiquitin proteasome system, such as PI31, Cdk6, p27, PINK1 (PTEN-induced kinase 1), and Parkin. PI31 is a known proteasome regulator which was initially characterized as a proteasome inhibitor in vitro. The binding affinity between Fbxo7 and PI31 is very strong, and The Fbxo7-PI31 interaction is mediated by heterodimerization of the FP domains of the two proteins. This work is focus on study the protein structure of the two FP domains in Fbxo7 and PI3. Chapter 1 reviewed the F-box-only protein Fbxo7 biology including the function of Fbxo7 protein in ubiquitination proteasome pathway and some SCF-independent functions which are relate to human disease. Chapter 2 discussed the function of proteasome inhibitor PI31. With the many important biological functions, Fbxo7 is clearly an extraordinary important protein, but the lack of structural knowledge has hampered efforts to achieve a better understanding of Fbxo7 biology. In this work, we have determined the crystal structure of Fbxo7 FP domain (residues 181-335) and the crystal structure of the PI31 FP domain (residues 1-161) using a longer protein construct both at 2.0Å resolution. The Fbxo7 FP domain adopts an α/β-fold similar to that of the PI31 FP domain and the secondary structure elements of the two FP domains are comparable including the C-terminal helix, indicating that the two FP domains share the same overall global fold. However, an α helix and three β strands in the Fbxo7 are longer than their counterparts in the PI31 FP domain. The two FP domains also differ substantially in the length and conformation of the longest connecting loop. More importantly, structural differences between the two FP domains lead to drastically different modes of inter-domain protein–protein interaction: the PI31 FP domain utilizes either an α interface or β interface for homodimeric interaction, whereas the Fbxo7 FP domain utilizes an αβ interface. We have note that the inter-domain interaction of the Fbxo7 FP domain is much more extensive, featuring a larger contact surface area, better shape complementarity and more hydrophobic and hydrogen-bonding interactions. The results of this structural study provide critical insights into how Fbxo7 may dimerize (or multimerize) and interact with PI31 via the FP domain. Chapter 4 and Chapter 5 discussed the structure determinations, structure features and detail of protein-protein interactions of Fbxo7 and PI31 FP domains. Chapter 2 reviewed the corresponding fundamental biochemical techniques that been used in this study. Chapter 3 discussed protein structure determination by X-ray crystallography in structural biology studies. It was believed that the FP domains of Fbxo7 and PI31 mediate homodimerization and heterodimerization of the proteins and the FP domain is not present in other human proteins. In order to study the Fbxo7-PI31 heterodimerization protein-protein interactions, we performed modeling studies. Chapter 6 discussed the model building and binding studies. Based on the result of model building studies, we propose that an interaction between the two FP domains of Fbxo7 and PI31 should be mediated by a αβ interface using the α-helical surface of the Fbxo7 FP domain and the β-sheet surface of the PI31 FP domain. According to the result of pull down assay, the PI31 FP domain may complete with Skp1 for the binding with Fbxo7. It is possible that the formation of heterodimer between the Fbxo7 and PI31 mediate by FP domains may lead to the Fbxo7 dissociation from SCFFbxo7 complex which might reveal a new regulation mechanism.

AN ABSTRACT OF THE THESIS OF BRENT C. SUNDERLAGE, for the Master of Science degree in Plant, Soil, and Agricultural Systems, presented on March 8, 2017, at Southern Illinois University Carbondale. TITLE: LATE APPLICATION NITROGEN ON CORN IN SOUTHERN ILLINOIS AND SOIL PROPERTIES AFFECTING AMMONIA VOLATILIZATION FROM UREA FERTILIZER MAJOR PROFESSOR: Dr. Rachel L. Cook In corn (Zea mays L.) production systems, typical nitrogen fertilization occurs either before planting or after crop emergence, as late as the V6 stage. Since the majority of nitrogen uptake does not occur until V10 through R1, delaying nitrogen fertilization until V10 may reduce potential for early season soil N transformations and losses, while sustaining crop yields. A two-year study, conducted across three southern Illinois locations, evaluated the effects of various late sidedress nitrogen applications and enhanced efficiency fertilizers on corn yield and residual mineral soil N. The various nitrogen treatments compared: rates from 0-224 kg N ha-1; sources of urea and 32% UAN, with and without urease inhibitors 0.09% N-(n-butyl) thiophosphoric triamide (NBPT) as Agrotain® Ultra (AT) and 0.06% NBPT + 0.02% N-(n-propyl) thiophosphoric triamide (NPPT) as Limus® (L), and polymer-coated urea (ESN®); application timings at planting, V6, V10, and VT; placement either dribble applied, broadcasted, or injected. Across sites and years, late (V10) nitrogen split applications generally resulted in corn yields greater than or equal to the same total nitrogen rate applied at planting. Most late N treatment differences varied between sites and years, but 56 kg N ha-1 as broadcasted UAN at planting with 112 kg N ha-1 as broadcasted urea, either with or without AT or L at V10 were on average highest yielding treatments among sites in both years, with 16% greater yield than 168 kg N ha-1 as broadcasted UAN at planting. Urease inhibitors did not enhance yield in most instances, likely due to sufficient incorporating rainfall shortly after application. The effects of urease inhibitors used with late-application nitrogen on corn yield and actual ammonia volatilization remained ambiguous. Residual soil mineral nitrogen concentrations between 0-30 cm after harvest in the late nitrogen treatments did not elicit water quality concerns. Furthermore, ammonia volatilization from surface applied urea is controlled by many interrelated soil properties as well as environmental conditions. However, conclusions about the influence of soil properties on ammonia volatilization differ according to geographies and are not well established across a wide range of soil types. A laboratory soil incubation experiment measured the effects of soil properties on ammonia volatilization over 7 days from surface-applied urea and the efficacy of three urease inhibitors: Agrotain® Ultra (AT) as 0.09% NBPT, Limus® (L) as 0.06% NBPT + 0.02% NPPT, and NutriSphere-N® (NS) 30% calcium salt of maleic-itaconic copolymer among 83 soil surface samples from across the United States with a wide range of soil properties. The soil properties evaluated were: total exchange capacity, 1:1 CaCl2 pH, organic matter, buffering capacity, clay content, and urease activity. In Urea (R2 = 0.69) and Urea + NS (R2 = 0.62) models, total exchange capacity, clay, and buffering capacity significantly reduced ammonia volatilization, and organic matter significantly increased ammonia volatilization, while 1:1 CaCl2 pH and urease activity were non-significant. Clay, organic matter, and buffering capacity were correlated to total exchange capacity. Total exchange capacity was the strongest predictor and best consolidated variable to predict N loss of urea. In Urea + AT (R2 = 0.54) and Urea + L (R2 = 0.67) models, ammonia volatilization was significantly reduced at lower 1:1 CaCl2 pH and total exchange capacity, and all other soil properties were non-significant. The NBPT in Urea + AT and Urea + L likely decayed more rapidly under acidic soil conditions, resulting reduced NBPT efficacy and greater N loss at lower pH. Urea + AT and Urea + L reduced volatilization significantly by 18.2 percentage points compared to Urea or Urea + NS, and there were no significant differences between Urea + AT and Urea + L (α = 0.05). On average, NutriSphere-N® did not reduce volatilization.