Student Number : 9600048X - MSc (Med) research report - Faculty of Health Sciences / Disorders of the thyroid gland are amongst the most common endocrine disorders. The diagnosis of thyroid disease consists of a history and clinical examination, followed by specific confirmatory investigations. These investigations are an important diagnostic component in thyroid disease and are amongst the most common investigations ordered in clinical laboratories. Although these tests are relatively inexpensive individually, they account for a disproportionately large amount of health care expenditure for diagnostic testing. Appropriate laboratory investigation is critical to establish the diagnosis and cause of thyroid disease in the most costeffective way. Discovery Health released a set of evidence-based guidelines in order to educate the clinician with regard to the selection of thyroid function tests. According to these guidelines a TSH (Thyroid Stimulating Hormone) test is the investigation of choice in suspected thyroid disease. This study is a retrospective investigation that compares the difference in ordering patterns of laboratory investigations by clinicians before and after the publication of the guidelines. Two data sets were generated from the data bank of Discovery Health. The first data set (I) was based on records compiled before March 2003 whereas the second data set (II) was based on records compiled from April 2003. Following use of the exclusion and inclusion criteria the sample size totaled 73 850 cases. An analysis was made with regard to the requesting frequency of specific tests. This study will focus solely on the appropriateness of thyroid function tests ordered. It is beyond the scope of this study to attach a specific clinical diagnosis to the results. The thyroid function tests requested before the publication of the evidence-based guidelines were often requested without careful thought and consideration on the part of the clinician. Some of the combination tests ordered (Free T3 and Free T4) are not advocated as an initial investigation in the evaluation of thyroid function and waste funds in this instance. The ordering of inappropriate thyroid function tests often leads to the depletion of funds available to a patient within the financial year. The results revealed that after publication of the guidelines there was an increase in the requesting frequency of TSH as a first line investigation, as well as Free T4 while a decrease in requests for Free T3 was noted. The publication of evidence-based guidelines as a guide to requesting the correct thyroid function tests in order to diagnose suspected thyroid disease appears to have impacted in increasing awareness amongst clinicians with regard to the tests required to diagnose and monitor thyroid disease.

Sköldkörteln (tyroidea) är ett endokrint organ som påverkar ämnesomsättningen. Tyreoidea stimulerande hormon (TSH) frisätts från hypofysen och stimulerar syntes och frisättning av tyreoideahormonerna trijodtyronin (T3) och tyroxin (T4). Tyreoidea hormonerna har i sin tur en negativ påverkan på frisättningen av TSH. TSH, T3 fritt och T4 fritt analyseras på serum med immunokemiska metoder. Becton Dickinson (BD) har tagit fram ett snabbkoagulerande provrör för analys av serum. Detta provrör heter BD Vacutainer® RST™ (rapid serum tube). Akutmottagningen på Centrallasarettet i Växjö använder detta provrör för vissa analyser och har önskemål om att kunna utföra tillbeställningar på S-TSH efter provtagning. Syftet med studien var att undersöka om det går att analysera S-TSH, S-T3 fritt och S-T4 fritt på BD Vacutainer® RST™ med instrumentet ADVIA Centaur XP. För studien togs venprover på 30 personer (20 kvinnor och 10 män), ett RST™ och ett SST™ (serum separation tube). SST™ användes som referensrör. Analysen av S-TSH, S-T3 fritt och S-T4 fritt utfördes med ADVIA Centaur XP och analysresultaten jämfördes genom regressionsanalys, Pearsons korrelationskoefficient och bias. Regressionsanalysen gav för alla tre analyserna linjer som låg nära varandra och k = 1, samt Pearsons korrelationskoefficient i närheten av 1. Bias visade en spridning på främst ±10 % med två avvikande resultat ett på 28 % och ett annat på -13 %. Dessa avvikande resultat förklarades genom att båda resultaten var låga värden och en liten skillnad mellan resultaten ger en större spridning. Slutsatsen blev att går det att analysera S-TSH, S-T3 fritt och S-T4 fritt på RST™ med ADVIA Centaur XP vilket kommer att minska antalet provtagningar på akuten och ge ett snabbare analysresultat på fler analyser.

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This thesis suggests the rudiments of a possible conceptual information system. Much of what is utilized in this effort is drawn from work completed and in progress. The system proposed is a synthesis and integration of divergent areas, in hopes of providing a better mode of comprehending the environmental process. The purpose of the thesis is to develop a system for handling data pertinent to environmental analysis within a conceptual framework. The thesis focuses on the initial conceptual information system analysis, which delineates the basic structure of the system. A conceptual framework proposed by Donald Foley is utilized as the beginning of the system. The conceptual framework consists of three levels (normative, functional-organizational, and physical), in which a further division is made by considering each level spatially and aspatially. Data is then stored within one of the six cells. A standard computer card format is suggested for data elements, and it is applicable to many varying situations. What is presented is not an all-inclusive system, but a basic framework that can receive additional increments of knowledge as they become available through time. Sample problems are injected to show how the system functions at various levels of concern. The passage of a sample problem through various components of the system by various agencies exhibits the system's ability to evaluate environmental phenomena. / M. Arch.

Este trabalho teve como principal objetivo a determinação dos parâmetros de soldagem a ponto pelo processo de fricção-mistura (FSSW) em uma liga de alumínio AA2024-T3, pela comparação de resultados obtidos em ensaios de cisalhamento e arrancamento em tração e ensaios de fadiga em juntas sobrepostas. Juntas rebitadas da mesma liga também foram ensaiadas, nas mesmas condições, para comparação de propriedades mecânicas. Os principais parâmetros do processo FSSW são: velocidades de avanço e de rotação da ferramenta, profundidade de penetração e tempo de patamar. Foram explorados principalmente os parâmetros: profundidade de penetração e rotação da ferramenta. Os resultados dos ensaios de cisalhamento em tração possibilitaram a determinação da resistência ao cisalhamento das juntas soldadas a ponto, que apresentaram valores inferiores, mas próximos aos obtidos nas juntas rebitadas. Análises microestruturais e de microdureza foram realizadas para elucidar alguns resultados dos ensaios executados. Os ensaios de fadiga foram executados sob controle de carga, com razão de carga R=0,1, em corpos de prova confeccionados com os parâmetros que geraram os melhores resultados em ensaios de cisalhamento em tração. Para as juntas soldadas, uma técnica de monitoramento de defeitos (MCV monitoramento comparativo de vácuo), baseada na diferença de pressão de vácuo, foi utilizada apresentando bons resultados. Apesar de resultados similares em ensaios de cisalhamento em tração, as juntas soldadas apresentaram vida bastante inferior nos ensaios de vida à fadiga, quando comparadas com as juntas rebitadas. / The main aim of this work was the determination of the parameters governing the Friction Stir Spot Welding (FSSW) of a AA2024-T3 aluminum alloy, by the obtained results comparison in pull-out and shear tensile tests and fatigue tests in lap-joints. Riveted joints of the same alloy were also tested in the same conditions for mechanical properties comparison. The main FSSW process parameters were: plunge rate, dwell time, tool penetration and tool rotational speed. The tool penetration and the tool rotational speed parameters were focused. The shear tensile tests results allowed the lap-joint shear resistance determination, that was lower, but close, of those obtained with riveted lap-joints. Microstructure and micro-hardness analysis was carried out to elucidate some tests perfomed results. The fatigue tests was performed in load control, with load ratio R of 0,1, in a lap joint produced with the same parameters of the specimen which presented the best tensile results. For the welded joints, a monitoring defect technique (CVM comparative vacuum monitoring), difference vacuum pressure based, was used showing good results. Although similar results in shear tensile tests, the welded joints showed so lower life in the fatigue tests, when compared with riveted joints.

Evidências sugerem interação entre o hormônio tireoideano (T3) e os fatores de crescimento fibroblásticos (FGF) no desenvolvimento esquelético. Para estudarmos essa interação, avaliamos o efeito do T3 e do seu antagonista NH3 em células mesenquimais periósticas humanas de pacientes normais e portadores da Síndrome de Apert (SA), que é caracterizada por craniossinostose e causada por mutações no receptor de FGF tipo 2 (FGFR2). Nas células SA, o T3 aumentou o número de células e o NH3 bloqueou esse efeito do T3. O T3 e/ou NH3 aumentaram a atividade da fosfatase alcalina durante a diferenciação osteoblástica das células normais, mas não das mutadas. O T3 aumentou a diferenciação osteoblástica e o NH3 bloqueou esse efeito do T3 em células normais. Nas células mutadas, o NH3 limitou a diferenciação osteoblástica, enquanto o T3 não teve efeito. Concluímos que as células mesenquimais periósticas humanas normais e SA são responsivas ao T3 e NH3, e que o T3 e FGF podem atuar através de vias de sinalização comuns na regulação da diferenciação osteoblástica. / Evidence suggests that there is an interaction between the thyroid hormone (T3) and fibroblast growth factors (FGFs) in the skeletal development. To study this interaction, we evaluated the effect of T3 and its antagonist, NH3, in human periosteal mesenchymal cells from normal and Apert Syndrome (AS) patients, which is characterized by craniosynostosis and is caused by mutations in FGF receptor type 2 (FGFR2). In AS cells, the T3 increased the number of cells and NH3 blocked this effect of T3. T3 and/or NH3 increased the alkaline phosphatase activity in osteoblast differentiation of normal cells, but not in the mutated cells. T3 increased osteoblast differentiation and NH3 blocked this effect of T3 on normal cells. In the mutated cells, NH3 limited osteoblast differentiation while T3 had no effect. We concluded that normal and AS human periosteal mesenchymal cells are responsive to T3 and NH3, and T3 and FGF may act through common signaling pathways in the regulation of osteoblastic differentiation.

Neste estudo foi avaliada a resistência à corrosão da liga 2024-T3 clad, anodizada em solução de ácido tartárico-sulfúrico (TSA) e pós-tratada em banhos contendo íons de cério (Ce(NO3)3) sem e com H2O2. O pós-tratamento visa aumentar a resistência à corrosão e conferir propriedades de auto regeneração à camada anodizada, sem, no entanto, causar o fechamento dos poros, mantendo assim as propriedades de adesão. Foram avaliados os efeitos da temperatura, do tempo de imersão e da porcentagem de H2O2 no banho de pós-tratamento sobre a microestrutura e resistência à corrosão. As observações por microscopia eletrônica de varredura (MEV) mostraram que não houve fechamento dos poros e que ocorre precipitação preferencial de oxihidróxidos de Ce nas proximidades dos defeitos da camada. A avaliação da resistência à corrosão em solução de NaCl 0,1 e 0,5 mol/L por espectroscopia de impedância eletroquímica (EIS) mostrou que a etapa de pós-tratamento em solução contendo 50 mM de Ce(NO3)3.6H2O e 10% vol. de H2O2 melhora o desempenho da camada, com o melhor resultado tendo sido obtido para a temperatura intermediária (50°C) de pós-tratamento. Os resultados dos ensaios de EIS mostraram também efeito negativo de altas temperaturas no pós-tratamento nas propriedades protetoras da camada anodizada. Esses resultados foram confirmados por fotos digitais e observação SEM das amostras após a conclusão dos experimentos de EIS. Já a caracterização composicional por meio de GDOES mostrou a incorporação de espécies de Ce dentro dos poros da camada anodizada após pós-tratamentos em solução contendo 50 mM de Ce(NO3)3.6H2O e 10% vol. H2O2 a 50°C para tempos de imersão de 2 e 5 minutos, resultados confirmados com a técnica RBS, que mostrou ainda a presença de Ce na superfície da camada e no interior dos poros mesmo após 15 dias de imersão em NaCl 0,1M. Medições de XPS mostraram que o Ce está presente na superfície das amostras nos estados de oxidação 3+ e 4+, e uma maior relação Ce3+/Ce4+ para as amostras pós-tratadas por 2 minutos. O ajuste dos diagramas de EIS com circuitos equivalentes mostrou que a selagem parcial dos poros tem papel importante na resistência à corrosão, e evidenciaram que os pós-tratamentos mais eficientes não afetam as propriedades protetoras da camada barreira. / This study evaluated the corrosion resistance of a 2024-T3 clad alloy anodized in tartaric-sulfuric acid solution (TSA) and post-treated in baths containing cerium ions, without or with H2O2. The post-treatment aims to increase the corrosion resistance and afford self-healing properties to the anodized layer, without, however, closing its porous structure, thus maintaining its adhesion properties. The effects of the temperature, immersion time and concentration of H2O2 in the post-treatment bath in the microstructure and in the corrosion resistance of the samples were evaluated. SEM observations showed that pores are not blocked and that preferential precipitation of Ce occurs in the vicinity of defective sites of the anodized layer. The evaluation of the corrosion resistance in 0.1 and 0.5 mol/L NaCl solution by means of electrochemical impedance spectroscopy (EIS) showed that the post-treatment in solutions containing 50 mM of Ce(NO3)3.6H2O and 10% vol. of H2O2 improves the corrosion resistance of the anodized samples, with the best result being obtained for the intermediary temperature (50°C) of the post-treatment bath. The results of the EIS tests also showed a negative effect of higher temperatures on the protective properties of the anodized layer. Digital photos and SEM observation of the samples after the completion of the EIS experiments confirmed these results. The compositional characterization by GDOES showed the incorporation of Ce species within the pores of the anodized layer after post-treatments in solution containing 50 mM Ce (NO3)3.6H2O and 10% vol. H2O2 at 50°C for 2 and 5 minutes, confirmed by the RBS technique, which also showed the presence of Ce both at the surface and within the pores of the layer, even after 15 days of immersion in 0.1M NaCl. XPS measurements showed the presence of Ce3+ and Ce4+ species at the post-treated samples surface and showed higher Ce3+ / Ce4+ ratio for the sample post-treated for 2 minutes. Fitting of the EIS diagrams with equivalent circuits showed that partial pore sealing plays an important role in corrosion resistance and evidenced that the most efficient post-treatments do not affect the protective properties of the barrier layer.

Os hormônios tireoidianos (HTs) desempenham um papel importante em diversos processos, tais como o crescimento, desenvolvimento e metabolismo dos tecidos em geral. Embora estudos tenham demonstrado que os HTs agem diretamente nas células foliculares da tireoide reduzindo sua resposta ao TSH, pouco se sabe, a nível molecular, sobre essa e outras ações dos HTs na própria glândula tireoide. Nesse sentido, o objetivo do presente trabalho foi avaliar alterações no perfil de expressão gênica nas células foliculares tireoidianas (células PCCl3) em reposta ao tratamento com triiodotironina (T3). Após atingir confluência desejada, as células PCCl3 foram mantidas em meio depletado de HTs (grupo Hipo) por 24h. Após esse período, parte das células foi tratada com 10-7M de T3 (grupo T3) por 24 h. As células foram, então, lisadas para extração de RNA total para análise do transcriptoma, por RNAseq. Foi obtido como resultado, uma lista de genes diferencialmente expressos da qual foram selecionados cinco genes para validação in vitro (novo lote de células PCCl3 submetidas às mesmas condições descritas acima) e in vivo [ratos Wistar (250g), tratados por 4 semanas com T3 (1,5 μg/100g de peso corporeo; grupo hipertireoideo) ou PTU (10 μg/100g de peso corporeo; grupo hipotireoideo): o Slc16a1, que codifica o MCT8, responsável pelo transporte de T3 através da membrana, o Snrpd1, 9-March, Pfdn1 e Fam103a1, que codificam proteínas envolvidas no controle pós-transcricional e pós-traducional da expressão gênica. O tratamento com T3 estimulou a expressão dos genes Snrpd1, Pfdn1 e Fam103a1, enquanto reduziu a expressão de 9-March e Slc16a1. Juntos esses resultados demonstram a existência de um efeito autócrino exercido pelo T3 sobre o controle do seu próprio turnover proteico. / Thyroid hormones (HTs) play an important role in many processes, such as growth, development and metabolism of tissues in general. Although studies have shown that HTs act directly on follicular thyroid cells reducing their response to TSH, little is known, at the molecular level, about this and other actions of HTs in the thyroid gland itself. In this sense, the aim of the present study was to evaluate changes in the gene expression profile in the thyroid follicular cells (PCCl3 cells) in response to triiodothyronine (T3) treatment. After reaching 60% confluence, PCCl3 cells were maintained in HT depleted medium (Hypo group) for 24h. After this time, part of the cells was treated with 10-7M T3 (T3 group) for 24 h. Cells were then lysed for total RNA extraction for transcriptome analysis by RNAseq. As a result, a list of differentially expressed genes from which five genes for in vitro validation (PCCl3 cells under the same conditions described above) and in vivo [Wistar rats (250g), treated for 4 weeks with T3 (1.5 &um;g / 100 g body weight, hyperthyroid group) or PTU (10 &um;g / 100 g body weight; hypothyroid group): Slc16a1, which encodes MCT8, responsible for the transport of T3 through the membrane, Snrpd1, 9-March, Pfdn1 and Fam103a1, which encode proteins involved in post-transcriptional and post-translational control of gene expression. T3 treatment stimulated the expression of the Snrpd1, Pfdn1 and Fam103a1 genes, while reducing the expression of 9-March and Slc16a1. Together these results demonstrate the existence of an autocrine effect exerted by T3 on the control of its own protein turnover.

Ziel dieser Arbeit war die Entwicklung neuer Substanzen für die Gentherapie. Diese beinhaltet die Behebung von erblich bedingten Krankheiten wie z.B. Mucoviscidose. Dabei werden im Zellkern defekte Gene durch normale, gesunde DNA-Sequenzen ersetzt. Zur Einschleusung des Genmaterials in die Zellen (Transfektion) werden geeignete Transport-Systeme bzw. Methoden benötigt, die dort die Freisetzung der neu einzubauenden Gene (Genexpression ausgedrückt in Transfektionseffizienzen) gestatten. Hierfür wurden neue Polykation-DNA-Komplexe (Vektoren) auf Basis kationischer Polymere wie Poly(ethylenimin) (PEI) hergestellt, charakterisiert und nachfolgend in Transfektionsversuchen an verschiedenen Zelllinien eingesetzt.<br> Sowohl das kationische Ausgangspolymer PEI als auch das Pfropfcopolymer PEI-g-PEO (PEO-Seitenketten zur Erhöhung der Biokompatibilität) wurden mit Rezeptorliganden modifiziert, um eine verbesserte und spezifische Transfektion an ausgesuchten Zellen zu erreichen. Als Liganden wurden Folsäure (Transfektion an HeLa-Zellen), Triiod-L-thyronin (HepG2-Zellen) und die Uronsäuren der Galactose, Mannose, Glucose sowie die Lactobionsäure (HeLa-, HepG2- und 16HBE-Zellen) verwendet.<br> Das PEI, die Pfropfcopolymere PEI-g-PEO und die Ligand-funktionalisierten Copolymere wurden hinsichtlich ihrer chemischen Zusammensetzung und molekularen Parameter charakterisiert. Die Molmassenuntersuchungen mittels Größenausschlusschromatographie zeigten, dass nach der Synthese unterschiedliche Polymerfraktionen mit nicht einheitlicher chemischer Zusammensetzung vorlagen.<br> Die anschließenden Transfektionsversuche wurden mit Hilfe einer speziellen DNA (Luciferase) an den Zelllinien HepG2 (Leberkrebszellen), HeLa (Gebärmutterhalskrebszellen) und 16HBE (Atemwegsepithelzellen) durchgeführt. Die T3(Triiod-L-thyronin)-Vektoren zeigten in Abhängigkeit vom eingesetzten Komplexverhältnis Polykation/DNA ein Maximum in der Transfektion an HepG2-Zellen. Die Hypothese der rezeptorvermittelten Endozytose ließ sich durch entsprechende T3-Überschuss-Experimente und Fluoreszenzmikroskopie-Untersuchungen bestätigen. Dagegen konnte bei den Folsäure-Vektoren keine rezeptorvermittelte Endozytose beobachtet werden.<br> Bei den Vektoren mit Mannuronsäure-Ligand (Man) konnte an allen drei Zelllinien (HepG2, HeLa, 16HBE) eine konstante, hohe Transfereffizienz nachgewiesen werden. Sie waren bei allen eingesetzten Polymer-DNA-Verhältnissen effizienter als der Vergleichsvektor PEI. Dieses Transfektionsverhalten ließ sich durch Blockierung der Zuckerstruktur unterbinden. In Transfektionsexperimenten mit einem Überschuss an freier Mannuronsäure und fluoreszenzmikroskopischen Untersuchungen konnte eine rezeptorvermittelte Endozytose der Man-Vektoren an den o.g. Zelllinien nachgewiesen werden. Die anderen Uronsäure-Konjugate zeigten keine signifikanten Abweichungen im Transfektionsverhalten im Vergleich zum PEI-Vektor. / The goal of this work was the development of new non-viral gene transfer systems for the somatic gene therapy. For these non-viral gene vectors (polycation-DNA-complexes) on the base of ligand-functionalized polycations were synthesized, characterized and tested in transfection trials on different cell cultures (HepG2, HeLa, 16HBE).<br> In preliminary investigations PEI-g-PEO copolymers with different grafting densities of poly(ethylene oxide) PEO8 were synthesized and characterized. This was followed by modification of PEI and the copolymer PEI-g-PEO(20) with specific receptor ligands for transfection studies to the cell lines mentioned above. Folic acid (transfection at HeLa cells), triiodo-L-thyronine (HepG2 cells) and the uronic acids of galactose, mannose, glucose as well as the lactobionic acid (HeLa, HepG2 and 16HBE cells) were used as ligands. The coupling of the ligands was performed either without a spacer or via PEO side chains and was realized by carbodiimids.<br> The PEI and the grafted copolymers PEI-g-PEO as well as the ligand-functionalized copolymers were characterized regarding to their chemical composition and molecular parameters. Molar masses from sedimentation rate experiments of the AUC were obtained within the range of 35000 to 70000 g/mol. The molar mass investigations by means of SEC-MALLS revealed that after the grafting process both copolymers with heterogeneous chemical composition and unmodified PEI were present. The polydispersity of all PEI-g-PEO(20) based copolymers increased significantly compared with unmodified PEI. The molar masses increased with higher conversion degree as expected. The highly-substituted products exhibited an increasingly more compact structure in aqueous solution.<br> The following transfection studies were accomplished with the help of a luciferase reporter genes at the cultures HepG2 (liver cancer cells), HeLa (cervix cancer cells) and 16HBE (lung epithelium cells). The grafted copolymers PEI-g-PEO were compared to the unmodified PEI vector in transfection experiments. Here an almost identical transfer efficiency compared to the unmodified PEI vector could be maintained accompanied by reduced toxicity up to a PEO content of 17% w/w.<br> Folic acid copolymers were tested on HeLa cells in further vector studies. All folic acid vectors showed a maximum in the transfection at a N/P ratio (complex of polycation with DNA) of 2.5 and/or 5.0, which refers to a receptor-mediated endocytosis. However, no receptor-mediated endocytosis was observed in transfection.<br> A similar transfection behavior was observed with the T3 vectors on HepG2 cells dependent on the N/P ratio. The hypothetical receptor-mediated endocytosis could be confirmed within the T3-functionalized vectors by appropriate T3 excess experiments. Herein the transfer efficiency of the T3 gene vectors decreased significantly while adding free low-molecular weight triiodo-L-thyronine. In contrast to this the transfer efficiency of the unmodified PEI vector decreased only negligibly. A receptor-mediated endocytosis was also confirmed by fluorescence microscopy investigation of T3-functionalized aminodextranes at transfection of HepG2 cells. Subsequently, the T3 vectors were tested at mice in vivo. Here high transfer efficiencies in comparison to the unmodified PEI vector were determined particularly in the spleen as well as in the kidneys and thyroid. The T3 vectors should be suitable for a gene transfer into hepatocytes.<br> The vectors with uronic acid conjugates as ligands (galacturonic-, glucuronic and lactobionic acid) did not show significant deviations in the transfer efficiencies compared with the PEI vector. In contrast to this the vectors with mannuronic acid exhibit a constant high transfer efficiency at the three cell cultures HepG2, HeLa and 16HBE. They are more efficient than the PEI vector over the examined N/P range. Here the transfection proceeds independently of the charge of the complex (N/P ratio). This transfection behavior could be prevented by blocking the glycosidic OH groups of the Man vector. A receptor-mediated endocytosis of the Man vectors at the three examined cell lines (HepG2, HeLa, 16HBE) could be verified by means of transfection experiments with an excess of free mannuronic acid and fluorescence microscopic investigations.<br> In continuing studies new gene vectors on the base of cationic starch graft copolymers were synthesized and tested in transfection studies at HepG2 and 16HBE cells. Beyond that peptide-functionalized PEI vectors, which exhibit a nuclear localization sequence (TAT), were established and their transfection in vitro was determined. Compared to the PEI vector lower transfections of the vectors on the base of cationic starch graft copolymers was observed. However, an increase is expected by coupling with T3 and mannuronic acid ligands.