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Optimization of immunotherapeutic relevant ABD-derived affinity proteins for prolonged serum half-lifeBergström, Ebba January 2022 (has links)
Marknaden för proteinbaserade läkemedel, de så kallade biologiska läkemedlen, är idag en industri som omsätter miljarder. Ett vanligt sätt att utveckla dessa läkemedel på är med hjälp av monoklonala antikroppar då de kan binda till sitt mål med hög specificitet. Däremot begränsas denna teknik av en lång och dyr produktion som dessutom kräver däggdjursbaserade uttrycksystem. En alternativ teknik till de monoklonala antikropparna är att använda små proteiner som enkelt kan produceras i bakterier till en låg kostnad. Dock begränsas denna metod av de små proteinernas korta cirkuleringstid i blodet. I ett tidigare projekt, har ett litet protein vid namnet ABDderived affinity ProTein (ADAPT) på cirka 7 kDa, utvecklats för att kunna binda till både humant serumalbumin (HSA) för att förlänga cirkulationstiden i blodet och Interleukin 17c (IL17c) som är ett pro-inflammatorisk cytokin. Studien visade dock att ADAPT proteinet inte samtidigt kunde binda till de båda molekylerna tillräckligt effektivt. Syftet med denna uppsats är därför att undersöka om det nämnda proteinet kan optimeras genom så kallad multimering och/eller manipulering av bindningssätet för HSA i syfte att åstadkomma en effektiv och mer långvarig cirkulationstid i blodet samtidigt som det binder sig till sitt mål, IL17c. Tio nya versioner av ADAPT proteinet har utvecklats genom att klona och transformera proteiner till en högt producerande Escherichia coli (E. coli) stam. Proteinerna har sedan producerats och renats fram. Det kunde observeras att proteinerna hade den önskade renheten för att kunna karaktäriseras. Vidare var det möjligt att se att proteinerna hade sin önskade molekylvikt och erhöll sin förväntade struktur som en alfahelix. Proteinernas smältpunkter hade förbättrats eller var liknande jämfört med det ursprungliga proteinet. Dessutom kunde alla proteiner återgå till sin ursprungliga struktur efter upphettning. Utvärderingen av proteinernas bindningskapacitet, med original proteinet som referens, visade på en ökad affinitet till sitt mål, IL17c, för två dimerer och trimeren samt en jämförbar affinitet för två av monomererna med ett manipulerat bindingssäte till HSA. Interaktion till HSA var jämförbar med den ursprungliga ADAPT molekylen för alla nya varianter förutom monomererna med ett manipulerat bindingssäte och dimeren med två manipulerat bindingssäten till HSA. Evaluering av de nya proteinernas kapacitet att binda samtidigt till HSA och IL17c visade att det var gynnsamt med en dimereiserad molekyl då det skapade en distans mellan molekylerna och dess bindningssäten. Vidare kunde det också visas att ordningen som molekylerna interagerade med varandra påverkade proteinernas simultana bindning. / The market for protein-based drugs, or the so-called biopharmaceuticals, is a multibillion-dollar industry today. In the development of protein-based drugs it is common to use monoclonal antibodies (mAbs) due to their ability to bind to its target with high specificity. However, therapeutical development of mAbs is limited by its long and expensive production in mammalian expression system. An alternative to mAbs are the so-called alternative scaffolds which are small proteins that can be produced in bacteria at lower costs. Although a drawback with the latter proteins is their short serum half-life. A small scaffold protein, ABD-Derived Affinity ProTein (ADAPT) of approximate 7 kDa was earlier engineered to obtain bispecific affinity, to Human Serum Albumin (HSA), to extend its half-life, as well as to the pro-inflammatory cytokine, Interleukin 17c (IL17c). Unfortunately, it was shown that the simultaneous binding was not efficient enough for its desired purpose. The aim with this project was therefore to investigate if the previous mentioned binder could be optimized by multimerization and/or manipulation of the HSA binding site for an efficient half-life extension. By generating ten new designs of the ADAPT variants, it was observed that the new variants had stable alpha helical structures and an improved or similar melting temperature as the original variant. The evaluation of the target binding displayed an improved affinity to the target, IL17c, for two of the dimeric versions as well as for the trimer and a comparable affinity for two of the monomers with a manipulated HAS binding site. The interaction to HSA was comparable to the original ADAPT for all binders except from the monomers with impaired HSA binding and the dimer with two impaired HSA binding sites. The evaluation of the simultaneous binding showed that it was favored by dimerization when a distance between the two molecule and their binding surfaces was added. Moreover, it could also be seen that the order of binding events had an impact on the simultaneous binding.
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Highly Efficient One-Step Protein Immobilization on Polymer Membranes Supported by Response Surface MethodologySchmidt, Martin, Abdul Latif, Amira, Prager, Andrea, Gläser, Roger, Schulze, Agnes 03 April 2023 (has links)
Immobilization of proteins by covalent coupling to polymeric materials offers numerous
excellent advantages for various applications, however, it is usually limited by coupling
strategies, which are often too expensive or complex. In this study, an electron-beambased
process for covalent coupling of the model protein bovine serum albumin (BSA)
onto polyvinylidene fluoride (PVDF) flat sheet membranes was investigated. Immobilization
can be performed in a clean, fast, and continuous mode of operation without any additional
chemicals involved. Using the Design of Experiments (DoE) approach, nine process factors
were investigated for their influence on graft yield and homogeneity. The parameters could
be reduced to only four highly significant factors: BSA concentration, impregnation
method, impregnation time, and electron beam irradiation dose. Subsequently,
optimization of the process was performed using the Response Surface Methodology
(RSM). A one-step method was developed, resulting in a high BSA grafting yield of
955 mgm−2 and a relative standard deviation of 3.6%. High efficiency was demonstrated
by reusing the impregnation solution five times consecutively without reducing the final
BSA grafting yield. Comprehensive characterization was conducted by X-ray
photoelectron spectroscopy (XPS), Fourier-transform infrared spectroscopy (FTIR), and
measurements of zeta potential, contact angle and surface free energy, as well as filtration
performance. In addition, mechanical properties and morphology were examined using
mercury porosimetry, tensile testing, and scanning electron microscopy (SEM).
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Dynamic interplay between activators and repressors of smooth muscle alpha-actin gene transcription during myofibroblast differentiationHariharan, Seethalakshmi 19 August 2014 (has links)
No description available.
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Apelin Regulation of K-Cl Cotransport in Vascular Smooth Muscle Cells.Sharma, Neelima 11 June 2014 (has links)
No description available.
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Inhibition of monoamine oxidase by derivatives of piperine, an alkaloid from the pepper plant Piper nigrum, for possible use in Parkinson’s diseaseAl-Baghdadi, Osamah Basim Khalaf 27 October 2014 (has links)
No description available.
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Characterization of Cys-34 in serum albuminTong, Grace C. 16 October 2003 (has links)
No description available.
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Post-Acute Serological Response to SARS-COV-2 and Predicting Post COVID-19 Condition (PCC) in CanadaCollins, Erin 05 January 2024 (has links)
Background: Post COVID-19 Condition (PCC, also known as long COVID and post-acute sequelae of COVID-19) is a major public health concern with severe and pervasive impacts on physical and mental health. PCC is highly heterogeneous and may manifest as different clusters of symptoms of varying intensity and duration. The etiology of PCC remains uncertain, though several underlying pathophysiological mechanisms, such as cellular damage, inflammatory cytokines, and a hypercoagulable state, are thought to contribute to PCC inception and trajectory. Examination of potential serological markers of PCC, accounting for clinical covariates, may yield emergent pathophysiological insights.
Objectives: Primary objectives of this thesis are to 1) Identify key clinical and potential serological predictors of PCC; 2) Acquire clinical and serological data in a large-scale prospective observational study; 3) Assess relationships between PCC and serological markers, accounting for clinical covariates; 4) Systematically review evidence to date on primary observational studies comparing serological response between people with and without persistent symptoms post COVID-19 recovery; 5) Discuss persisting gaps in knowledge and data quality, and propose strategies for resolve.
Methods: This thesis is framed around three core efforts: 1) The design of survey questions and study materials, recruitment of participants, and data collection in a large-scale prospective cohort study launched in 2020; 2) The assessment of relationships between pre-defined serological predictors and PCC, accounting for clinical covariates; and 3) A robust rapid review of PCC onset and phenotype as functions of serological markers. Expert opinion was sought to define serological predictors. Clinical predictors were defined a priori based on systematic reviews meeting AMSTAR 2 guidelines.
Conclusions: To address objectives, we described efforts to collect clinical and serological data from a large-scale prospective cohort study; identify PCC-cases and infected-controls; assess associations between pre-defined serological predictors (IgG titres targeting SARS-CoV-2 spike (S), nucleocapsid (N), and receiver binding domain (RBD) antigens, and efficient neutralization) and PCC; and synthesized findings from an extensive rapid review on PCC as a function of serological markers. Our multivariate analysis using Stop the Spread Ottawa data is, to our knowledge, the first Canadian study to report the direction and magnitude of association between selected serological predictors (anti-IgG response to S, N, and RBD SARS-CoV-2 antigens, and neutralizing efficiency) and PCC status and impact on quality of life. Finally, we described five potential strategies which may improve the accessibility, quality, and amalgamation of data pertaining to PCC: 1) Fostering comparability between studies to enable synthesis of multiple datasets; 2) Advancing the characterization and consensus on PCC phenotypes; 3) Employing innovative modelling strategies that could potentially yield novel insights; 4) Promoting robust collaboration and knowledge sharing among research teams; and 5) Engaging people with lived experience at all stages of research.
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PKN1 is a novel therapeutic target to block serum response factor-dependent androgen receptor action in advanced prostate cancer.Venkadakrishnan, Varadha Balaji 30 September 2020 (has links)
No description available.
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Exercise, Fatigue and Serum Inflammatory Cytokine Changes in People with Relapse Remitting Multiple Sclerosis: A Pilot StudyXiong, Jin Li January 2019 (has links)
Fatigue is a prevalent and debilitating symptom that affects up to 97% of individuals with multiple sclerosis (MS). It can negatively influence the socioeconomic status, activities of daily living and quality of life for the affected individuals. Fatigue is multidimensional and abstract, thereby making it complex to understand, target and treat. Over the past 20 years, physical activity has become more recognized as a management method that could help with the alleviation of fatigue. One of the reasons could be due to the anti-inflammatory effects of exercise. However, due to the multi-factorial nature of fatigue and the heterogeneity of the training intervention protocols, the potential mechanisms that underlie the relationship between fatigue and exercise are still not fully understood. In 2013, Latimer-Cheung and colleagues developed an evidence based physical activity guideline (PAGs) for people with MS. Since then, studies have shown consistent beneficial effects of exercise on reducing fatigue in people with MS by adhering to the PAGs. To date, however, there are no published studies that examined the potential mechanism that underlie the beneficial effect of the PAGs on reducing fatigue. The primary purpose of this thesis was to evaluate the effects of adhering to the PAGs on fatigue in people with MS and to assess whether any exercise-induced changes in fatigue were associated with changes in inflammatory cytokines. The secondary purpose of this thesis was to evaluate the effects of exercise on depression, strength, aerobic fitness, muscular endurance and quality of life. This study had a wait-list control design. Participants with relapse remitting multiple sclerosis (RRMS) were recruited and randomized to begin with either a 12-week supervised exercise training program (G1) or a wait-list control period (G2). The training program involved at least 30 minutes aerobic training and resistance training for major muscle groups twice per week. The G2 group maintained their regular lifestyle. After 12 weeks, G1 reverted back to their usual lifestyle and G2 began their 12-week supervised exercise training. Following training, we found a reduction in fatigue and depression with increased strength and quality of life. No changes were observed in pro-inflammatory cytokines, aerobic fitness or muscular endurance. This is the first study that examined the underlying potential mechanism for the beneficial effects of exercise by adhering to the PAGs. Following the PAGs for 12 weeks results in significant improvements in fatigue, depression, strength and quality of life. However, our results do not support the role of inflammatory cytokines in mediating these improvements. / Thesis / Master of Science in Kinesiology
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Produção de proteínas recombinantes em células BHK-21 cultivadas em meio livre de soro fetal bovino. / Production of recombinant proteins in BHK-21 cells cultured in serum free media.Patiño, Sandra Fernanda Suárez 06 May 2016 (has links)
Células eucariotas usadas como plataforma de expressão de proteínas recombinantes são geralmente cultivadas com soro fetal bovino (SFB), porém, abordagens biotecnológicas atuais sobre cultura de células devem evitar o uso deste suplemento, devido a problemas de custo, variações entre os lotes e risco de contaminação. Assim, nosso objetivo foi expressar as proteínas recombinantes: GFP (proteína verde fluorescente), NS3 (proteína não estrutural 3 do vírus da hepatite C) e RVGP (glicoproteína do vírus da raiva) em células BHK-21 adaptadas em meios livres de soro fetal bovino (SFM) usando o sistema de expressão baseado no Semliki Forest Virus (SFV). Os resultados do presente trabalho mostraram que células adaptadas em SFM cresceram de forma eficiente, produziram mais partículas virais recombinantes de SFV do que células suplementadas com soro, sendo que estas partículas virais podem ser usadas diretamente para imunização, pois garantiram uma amplificação e expressão eficiente das diferentes proteínas dentro da célula hospedeira. / Eukaryotic cells are cultured with serum, however current biotechnological approaches of cell culture need to avoid using of this supplement, due to the high costs, lot-to-lot variation and risk of contamination. Thus, our aim was to express the recombinant protein: GFP (green fluorescent protein); NS3 (Hepatitis C virus non-structural protein 3) and RVGP (rabies virus glycoprotein) in BHK-21 cells cultured in serum free culture based on Semliki Forest Virus system. The results of this work showed that cells cultured in serum-free media (SFM) were grown efficiently, they were produce more recombinant viral particles when compared with cells supplemented with SFB. These viral particles can be used directly for immunization, since generated amplification and expression efficient of different proteins within the host cell.
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