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Artykuły w czasopismach na temat „Human Biophysics”

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Autor: Grafiati

Data publikacji: 4 czerwca 2021

Data aktualizacji: 31 stycznia 2023

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1

Kalashnikov, Nikita, i Christopher Moraes. "Engineering physical microenvironments to study innate immune cell biophysics". APL Bioengineering 6, nr 3 (1.09.2022): 031504. http://dx.doi.org/10.1063/5.0098578.

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Innate immunity forms the core of the human body's defense system against infection, injury, and foreign objects. It aims to maintain homeostasis by promoting inflammation and then initiating tissue repair, but it can also lead to disease when dysregulated. Although innate immune cells respond to their physical microenvironment and carry out intrinsically mechanical actions such as migration and phagocytosis, we still do not have a complete biophysical description of innate immunity. Here, we review how engineering tools can be used to study innate immune cell biophysics. We first provide an overview of innate immunity from a biophysical perspective, review the biophysical factors that affect the innate immune system, and then explore innate immune cell biophysics in the context of migration, phagocytosis, and phenotype polarization. Throughout the review, we highlight how physical microenvironments can be designed to probe the innate immune system, discuss how biophysical insight gained from these studies can be used to generate a more comprehensive description of innate immunity, and briefly comment on how this insight could be used to develop mechanical immune biomarkers and immunomodulatory therapies.

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Stiers, Kyle M. "Personalized biophysics of human PGM1 deficiency". Acta Crystallographica Section A Foundations and Advances 73, a1 (26.05.2017): a167. http://dx.doi.org/10.1107/s0108767317098348.

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Yanaka, Saeko, Takamasa Ueno, Kouhei Tsumoto i Kenji Sugase. "Revealing the peptide presenting process of human leukocyte antigen through the analysis of fluctuation". BIOPHYSICS 11 (2015): 103–6. http://dx.doi.org/10.2142/biophysics.11.103.

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Connelly, Patrick R. "Recent drug discovery success signals renaissance in biophysics". Biophysics Reviews 3, nr 2 (czerwiec 2022): 020401. http://dx.doi.org/10.1063/5.0099305.

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With a scope that spans the hierarchy of biological organization from molecules and cells to organisms and populations, the discipline of biophysics has been proven to be particularly well suited for connecting the molecular embodiments of human diseases to the medical conditions experienced by patients. Recently, fundamental biophysical research on aberrant proteins involved in maintaining salt and water balance in our lungs, oxygen transport from our lungs to the rest of the body, and the pumping of blood by our hearts have been successfully translated to the creation of transformational new medicines that are radically changing the lives of patients. With these and other emerging discoveries, the field of applied biophysics is experiencing the beginnings of a veritable renaissance era.

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Vincze, Janos, Gabriella Vincze-Tiszay i Julianna Szakacs. "The Biophysical Modeling of the Hemodynamic in the Human Organism". Advances in Social Sciences Research Journal 7, nr 11 (8.12.2020): 494–502. http://dx.doi.org/10.14738/assrj.711.9420.

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The circulatory apparatus has as a main function the constant maintaining of the internal environment in all the regions of the organism. The blood is a liquid tissue, being formed of a fundamental substance – plasma and blood cells. Heart is the central organ of the cardiovascular apparatus. The heart muscles have numerous biophysical properties. The cardiac muscle is never tired unless it suffered a pathological process. During the diastole, blood is aspired in the heart and during the systole it is pushed in the big and small circulation. The blood amount pushed from the heart in the vascular system in a certain time represents the blood flow. The biophysical methods are next: we administer a certain substance amount, then its passing speed will depend on its concentration; to apply the calorimetric principles for the measurement of the gastric blood flow; the diagnostic of a chronic peripheral arteriopathy we use the calorimetric method is based on measuring the heat being introduced in a certain amount of water which has known temperature; one of the most often used methods for the evaluation of the use of radioisotopes in the cardio-vascular system is the compartment method. Any attempt to apply biophysics to the life systems involves three stages. First we observe the phenomena and formulate a biophysical description in the form of equations; after to solve the equations. Finally we return to the real life system and interpret this solution in terms of reality, this interpretation may requiew experimental testing.

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Sprinzak, David, i Stephen C. Blacklow. "Biophysics of Notch Signaling". Annual Review of Biophysics 50, nr 1 (6.05.2021): 157–89. http://dx.doi.org/10.1146/annurev-biophys-101920-082204.

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Notch signaling is a conserved system of communication between adjacent cells, influencing numerous cell fate decisions in the development of multicellular organisms. Aberrant signaling is also implicated in many human pathologies. At its core, Notch has a mechanotransduction module that decodes receptor–ligand engagement at the cell surface under force to permit proteolytic cleavage of the receptor, leading to the release of the Notch intracellular domain (NICD). NICD enters the nucleus and acts as a transcriptional effector to regulate expression of Notch-responsive genes. In this article, we review and integrate current understanding of the detailed molecular basis for Notch signal transduction, highlighting quantitative, structural, and dynamic features of this developmentally central signaling mechanism. We discuss the implications of this mechanistic understanding for the functionality of the signaling pathway in different molecular and cellular contexts.

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7

Summers, RL. "Physiology and Biophysics of the 100-m Sprint". Physiology 12, nr 3 (1.06.1997): 131–36. http://dx.doi.org/10.1152/physiologyonline.1997.12.3.131.

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The 100-m sprint is the most exciting of Olympic events, and the winner is often called the "world's fastest human." The physiology of this race is unique and invovles our most primordial survival mechanisms. A computer stimulation predicts the theoretical "physiological" limits for human achievement in this event.

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8

Lee, Keel Yong, Sung-Jin Park, David G. Matthews, Sean L. Kim, Carlos Antonio Marquez, John F. Zimmerman, Herdeline Ann M. Ardoña, Andre G. Kleber, George V. Lauder i Kevin Kit Parker. "An autonomously swimming biohybrid fish designed with human cardiac biophysics". Science 375, nr 6581 (11.02.2022): 639–47. http://dx.doi.org/10.1126/science.abh0474.

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Biohybrid systems have been developed to better understand the design principles and coordination mechanisms of biological systems. We consider whether two functional regulatory features of the heart—mechanoelectrical signaling and automaticity—could be transferred to a synthetic analog of another fluid transport system: a swimming fish. By leveraging cardiac mechanoelectrical signaling, we recreated reciprocal contraction and relaxation in a muscular bilayer construct where each contraction occurs automatically as a response to the stretching of an antagonistic muscle pair. Further, to entrain this closed-loop actuation cycle, we engineered an electrically autonomous pacing node, which enhanced spontaneous contraction. The biohybrid fish equipped with intrinsic control strategies demonstrated self-sustained body–caudal fin swimming, highlighting the role of feedback mechanisms in muscular pumps such as the heart and muscles.

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9

Kuo, A. D. "BIOPHYSICS: Harvesting Energy by Improving the Economy of Human Walking". Science 309, nr 5741 (9.09.2005): 1686–87. http://dx.doi.org/10.1126/science.1118058.

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Ernest, Nola Jean, Naomi J. Logsdon, Michael B. McFerrin, Harald Sontheimer i Susan E. Spiller. "Biophysical Properties of Human Medulloblastoma Cells". Journal of Membrane Biology 237, nr 2-3 (październik 2010): 59–69. http://dx.doi.org/10.1007/s00232-010-9306-x.

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Simon, S. I., i G. W. Schmid-Schönbein. "Biophysical aspects of microsphere engulfment by human neutrophils". Biophysical Journal 53, nr 2 (luty 1988): 163–73. http://dx.doi.org/10.1016/s0006-3495(88)83078-9.

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Kiemeneij, Wilbert, Lesley de Putter i Maaike Koopman. "Learning biophysics with open simulations". Physics Education 58, nr 2 (20.12.2022): 025006. http://dx.doi.org/10.1088/1361-6552/aca7f6.

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Abstract Physics education in secondary schools can include biophysics topics as electives. An open simulation based on a simulation like Minecraft for learning the workings of the action potential in human cells was designed and tested for students in upper secondary physics education. In small design and test cycles both the simulation engine and the classroom materials were developed. The simulation shows realistic results that go beyond the accompanying textbook. The finished prototype of the engine and the materials were used to study student reasoning during their simulation of the action potential. Results show that students reason together about what they see in the simulation to come to theories on the action potential process, showing profound learning. The students did not master the more open inquiry final assignment.

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13

Tabacchi, Marco Elio, i Settimo Termini. "“The human use of human beings”: Interdisciplinarity, transdisciplinarity and all that in biophysics and beyond". Biophysical Chemistry 229 (październik 2017): 165–72. http://dx.doi.org/10.1016/j.bpc.2017.05.007.

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14

Oshinowo, Oluwamayokun, Renee Copeland, Yumiko Sakurai, Meredith E. Fay, Brian G. Petrich, Traci Leong, Benjamin Brainard i Wilbur A. Lam. "Significant differences in single-platelet biophysics exist across species but attenuate during clot formation". Blood Advances 5, nr 2 (20.01.2021): 432–37. http://dx.doi.org/10.1182/bloodadvances.2020003755.

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Key Points Human, canine, ovine, and porcine platelets exhibit disparate biophysical signatures, whereas human and murine platelets are similar. Multiple biophysical parameters integrate during clot formation, measured by bulk clot contraction, and attenuate biophysical differences.

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15

Ji, Hong-Long, Run-Zhen Zhao, Zai-Xing Chen, Sreerama Shetty, Steven Idell i Sadis Matalon. "δ ENaC: a novel divergent amiloride-inhibitable sodium channel". American Journal of Physiology-Lung Cellular and Molecular Physiology 303, nr 12 (15.12.2012): L1013—L1026. http://dx.doi.org/10.1152/ajplung.00206.2012.

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The fourth subunit of the epithelial sodium channel, termed delta subunit (δ ENaC), was cloned in human and monkey. Increasing evidence shows that this unique subunit and its splice variants exhibit biophysical and pharmacological properties that are divergent from those of α ENaC channels. The widespread distribution of epithelial sodium channels in both epithelial and nonepithelial tissues implies a range of physiological functions. The altered expression of SCNN1D is associated with numerous pathological conditions. Genetic studies link SCNN1D deficiency with rare genetic diseases with developmental and functional disorders in the brain, heart, and respiratory systems. Here, we review the progress of research on δ ENaC in genomics, biophysics, proteomics, physiology, pharmacology, and clinical medicine.

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16

Sidar, Barkan, Jonathan N. V. Martinson, Jason R. Spence, Seth T. Walk i James N. Wilking. "Structuring Human Intestinal Organoids". Biophysical Journal 110, nr 3 (luty 2016): 168a. http://dx.doi.org/10.1016/j.bpj.2015.11.935.

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Klosterman, Katja E., Kiersten D. Lenz, Harshini Mukundan i Jessica Kubicek-Sutherland. "Biophysical Characterization of Human Lipoproteins for Diagnostic Assay Development". Biophysical Journal 120, nr 3 (luty 2021): 232a. http://dx.doi.org/10.1016/j.bpj.2020.11.1538.

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White, Natalie. "Overexpression and Biophysical Characterization of Human Interleukin-1 Alpha". Biophysical Journal 98, nr 3 (styczeń 2010): 499a. http://dx.doi.org/10.1016/j.bpj.2009.12.2719.

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19

Ramachandran, Jayalakshmi, Jorge E. Contreras i R. Daniel Peluffo. "Biophysical Properties of a Human Plasma-Membrane Creatine Transporter". Biophysical Journal 104, nr 2 (styczeń 2013): 108a. http://dx.doi.org/10.1016/j.bpj.2012.11.629.

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Ferrantini, Cecilia, Beatrice Scellini, Giulia Vitale, J. Manuel Pioner, Silvia Querceto, Raffaele Coppini, Nicoletta Piroddi, Corrado Poggesi i Chiara Tesi. "Mavacamten depresses human atrial contractility in the same EC50% range as human ventricle". Biophysical Journal 121, nr 3 (luty 2022): 106a—107a. http://dx.doi.org/10.1016/j.bpj.2021.11.2179.

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Laura, Patricio A. A. "Vibration analysis of human tibia". Journal of Biomedical Engineering 8, nr 3 (lipiec 1986): 278. http://dx.doi.org/10.1016/0141-5425(86)90098-1.

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Gopala Krishna, G., A. K. W. Anwar Ali, D. Ram Mohan i Adeel Ahmad. "Dielectrophoretic study of human erythrocytes". Journal of Biomedical Engineering 11, nr 5 (wrzesień 1989): 375–80. http://dx.doi.org/10.1016/0141-5425(89)90099-x.

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Brundin, J., A. Molin, S. Lundberg, C. Somnell i U. Ulmsten. "Measurement of human cervical compliance". Journal of Biomedical Engineering 11, nr 2 (marzec 1989): 148–50. http://dx.doi.org/10.1016/0141-5425(89)90126-x.

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Midic, U., C. J. Oldfield, A. K. Dunker, Z. Obradovic i V. N. Uversky. "Unfoldomics of Human Genetic Diseases". Biophysical Journal 96, nr 3 (luty 2009): 318a. http://dx.doi.org/10.1016/j.bpj.2008.12.1594.

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Berglund, Emily A., Jonathan N. V. Martinson, Jason R. Spence, Seth T. Walk i James N. Wilking. "Healing of Human Intestinal Organoids". Biophysical Journal 110, nr 3 (luty 2016): 171a. http://dx.doi.org/10.1016/j.bpj.2015.11.952.

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DuChez, Brian J., Andrew D. Doyle, Emilios K. Dimitriadis i Kenneth M. Yamada. "Durotaxis by Human Cancer Cells". Biophysical Journal 116, nr 4 (luty 2019): 670–83. http://dx.doi.org/10.1016/j.bpj.2019.01.009.

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Poulsen, Mette H., Svetlana R. Maurya, Johann Sigurdsson, Alicia Lundby i Stephan A. Pless. "Protein-Protein Interactions of Human P2X7 in Microglia and Human ASIC1a in Kidney Cells". Biophysical Journal 118, nr 3 (luty 2020): 584a. http://dx.doi.org/10.1016/j.bpj.2019.11.3167.

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Vincze, Janos, i Gabriella Vincze-Tiszay. "Some Stress Aspects in the Human Organism". Journal of Clinical and Laboratory Research 5, nr 4 (31.03.2022): 01–03. http://dx.doi.org/10.31579/2768-0487/077.

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In the human organism, the stress is a perturbation. The existence of the perturbations imposes a permanent control of the way the exit size is realized, so that any deviation can be corrected. Stress represents a normal reaction of the organism which appears as a response to an aggression situation which requires an unusual and quick adaptation effort from the organism. Stress is a state of putting in alert, of mobilizing the forces of the organism in the occasion of an event which requires, in order to be kept under control, a big amount of energy in a very short time. This alert state or action preparation translated through physical and psychological manifestations. Psychical stressors are those which come into being independently from the man’s will and destroy – as social factors – psychic and organic components taking place in the response, the organism consequently get tired. During the harmonic life gradually all human psycho-organic components shall get exhausted. During the individual evolution, the immediate vicinity imparts the codex of the principles of the moral motions. This is also a matter of opinion a kind of scale of values, more one can say it is very favourable for the individual from the healthy-spiritual point of view, because they are hardly touched by the negative effect of stress usually people beyond the pale of the society, in straits to the periphery. The physical discomfort feeling with the concomitant psychical reaction is realized by the measure of the personality’s sensitiveness. The modeling method in biophysics consists in the negative inverse connection

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Copeland, Renee, Oluwamayokun T. Oshinowo, Yumiko Sakurai, Meredith Ellen Fay, Brian G. Petrich, Benjamin Brainard, Wilbur A. Lam i David R. Myers. "A Comparative Medicine Study of Platelet Biophysics Among Hemostasis Models of Different Species". Blood 132, Supplement 1 (29.11.2018): 869. http://dx.doi.org/10.1182/blood-2018-99-113107.

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Abstract Background: Animal models of hemostasis play a crucial role in understanding hemostasis and developing novel therapeutic treatments. Hemostasis is a mechanical phenomenon and recent studies have demonstrated that biophysical parameters, such as platelet margination (Walton et al. Blood 2017), thrombus porosity (Welsh et al. Blood 2014), shear forces (Nesbitt et al. Nat Med 2009), compression forces (Ju et al. Nat Comm 2018), and single platelet forces, as measured by platelet contraction cytometry, developed by our laboratory, (Myers et al. Nat Mat. 2017) affect hemostatic processes and may be pathologically altered in disease states. However, little is known about the most basic biophysical interspecies differences between platelets of various animal hemostasis models, which could provide critical insight into interpreting data and results. Here, we report the results of our initial study on platelet biophysics in humans (n=5), mice (n=8), pigs (n=5), and dogs (n=6), which are commonly used for hemostasis models due to anatomical similarities to human vessels, organs, and cell function (Siller-Matula et al. Thromb & Haem 2008) (Jagadeeswaran et al. Circ Res 2015). We focus on understanding differences in platelet adhesion on collagen and fibrinogen, spreading area, singe cell contraction forces, and bulk contraction, which all may influence the initiation and stability of blood clots (Fig. 1). Our data demonstrates striking differences between each species, especially concerning adhesion, spreading area, and single platelet force. Surprisingly, measurements of volumetric bulk contraction, the most commonly used biophysical assay in hemostasis research, appear to be quite consistent between species. Methods: We performed a comprehensive study of platelet biophysics using adhesion assays, bulk contraction, and single platelet contractile forces on humans, mice, pigs, and dogs' platelets. For the adhesion assays, spreading area, and platelet contractile forces, platelets were gel-filtered as described previously (Myers et al. 2017). Cell Adhesion & Spreading Assay: Gel filtered platelets were diluted to 20M/mL, incubated on coverslips that had been incubated with type 1 collagen (100 µg/mL) or fibrinogen (100 µg/mL), allowed to adhere for 2 hours, counted, and measured. Platelet Contraction Cytometry: Polyacrylamide hydrogels with pairs of fibrinogen microdots are fabricated at physiologically relevant stiffnesses. Gel-filtered thrombin-activated platelets (1 U/mL) are then plated to attach, spread, and contract the microdots together. Individual platelet force is directly proportional to the microdot displacement measured with fluorescent imaging. Bulk Clot Contraction Assay: Platelets are washed as described previously (Lam et al. Nat Mat 2011) and then diluted to 200M/mL in a solution of fibrinogen (2 mg/mL), CaCl2 (5 mM), and thrombin (1 U/mL). Clots are left to form in cuvettes lined with gridded paper and measured every 30 mins for 2.5 hours. All assays were performed on at least 3 subjects from each species and in triplicate when possible. Results: We found both significant biophysical differences and similarities between platelets of different species (Fig. 2). Overall, murine platelets have the highest biophysical similarity to human platelets regarding adhesion, volumetric contraction, and single platelet contractile force despite substantial size differences. Porcine platelets exhibited enhanced adhesion on collagen with little adhesion to fibrinogen; while canine platelets showed the opposite. Such differences could have significant implications on the biophysical initiation of blood hemostasis and thrombosis, as well as the thrombogenicity tests of new materials. In bulk and single platelet contraction, we noted that similarities between most species except for canine platelets which produced forces nearly triple that of humans, the highest contractile forces of any platelets measured to date. As recent work has shown that forces are linked to clot porosity and therefore activation (Mirramezani et al. JTH 2018), such high forces could influence the clot hierarchical structure and subsequent lysis. Taken together, these results demonstrate how the biophysical properties of platelets differ across species and may be another reason why animal studies of hemostasis do not always translate to humans. Disclosures No relevant conflicts of interest to declare.

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Paulus, Walter, i John C. Rothwell. "Membrane resistance and shunting inhibition: where biophysics meets state-dependent human neurophysiology". Journal of Physiology 594, nr 10 (12.05.2016): 2719–28. http://dx.doi.org/10.1113/jp271452.

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Froment, Carine, Clément Zanolli, Mathilde Hourset, Emmanuelle Mouton-Barbosa, Andreia Moreira, Odile Burlet-Schiltz i Catherine Mollereau. "Protein sequence comparison of human and non-human primate tooth proteomes". Journal of Proteomics 231 (styczeń 2021): 104045. http://dx.doi.org/10.1016/j.jprot.2020.104045.

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Daniels, Brian R., Christopher M. Hale, Shyam B. Khatau, Sravanti Kusuma, Terrence M. Dobrowsky, Sharon Gerecht i Denis Wirtz. "Differences in the Microrheology of Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells". Biophysical Journal 99, nr 11 (grudzień 2010): 3563–70. http://dx.doi.org/10.1016/j.bpj.2010.10.007.

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Mikhalchik, E. V., N. V. Smolina, T. S. Astamirova, I. V. Gorudko, D. V. Grigorieva, V. A. Ivanov, A. V. Sokolov, V. A. Kostevich, S. N. Cherenkevich i O. M. Panasenko. "Human serum albumin modified under oxidative/halogenative stress enhances luminol-dependent chemiluminescence of human neutrophils". Biophysics 58, nr 4 (lipiec 2013): 530–36. http://dx.doi.org/10.1134/s0006350913040118.

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Pedersen, Morten Gram. "A Biophysical Model of Electrical Activity in Human β-Cells". Biophysical Journal 99, nr 10 (listopad 2010): 3200–3207. http://dx.doi.org/10.1016/j.bpj.2010.09.004.

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Volobuev, V., A. Kolsanov, N. Romanchuk, D. Romanov, I. Davydkin i Pyatin. "Genetic-Mathematical Modeling of Population Interaction, New Psychoneuroimmunoendocrinology and Psychoneuroimmunology". Bulletin of Science and Practice 6, nr 11 (15.11.2020): 85–103. http://dx.doi.org/10.33619/10.33619/2414-2948/60/09.

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Modern digital healthcare, biophysics and biology create new problems that stimulate the development of a new biophysical circuit and mathematical models from nuclear fusion (nuclear medicine) to genomic cell-organizational prognosis in neurophysiology, neuroendocrinology, psychoneuroimmunology and psychoneuroimmunoendocrinology. In this case, the following are effectively used: deterministic, stochastic, hybrid, multiscale modeling methods, as well as analytical and computational methods. The solution of a genetical-mathematical problem of interaction of cages of human population and virus population in relation to COVID-19 pandemic problem is submitted. A mathematical model based on the Hardy-Weinberg law is used, consisting of two interdependent differential equations. The equations reflect the temporal dynamics of the cells of human and viral populations during their interaction. Solutions of differential equations were found and the results of these solutions were analyzed. The pandemic duration is estimated using parameters of human liver cells and influenza virus. Perspective of further development of psychoneuroimmunology as interdisciplinary science, through algorithms and routing of digital health care, with expansion of psychoneurocommunications of professional interests in medicine, economics, sociology, cultural studies is shown. Modern neurobes and neuromarketing are built around Homo sapiens within a “reasonable environment” — a healthy individual space.

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Fuentes, Agustin. "Human niche, human behaviour, human nature". Interface Focus 7, nr 5 (18.08.2017): 20160136. http://dx.doi.org/10.1098/rsfs.2016.0136.

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The concept of a ‘human nature’ or ‘human natures’ retains a central role in theorizing about the human experience. In Homo sapiens it is clear that we have a suite of capacities generated via our evolutionary past, and present, and a flexible capacity to create and sustain particular kinds of cultures and to be shaped by them. Regardless of whether we label these capacities ‘human natures’ or not, humans occupy a distinctive niche and an evolutionary approach to examining it is critical. At present we are faced with a few different narratives as to exactly what such an evolutionary approach entails. There is a need for a robust and dynamic theoretical toolkit in order to develop a richer, and more nuanced, understanding of the cognitively sophisticated genus Homo and the diverse sorts of niches humans constructed and occupied across the Pleistocene, Holocene, and into the Anthropocene. Here I review current evolutionary approaches to ‘human nature’, arguing that we benefit from re-framing our investigations via the concept of the human niche and in the context of the extended evolutionary synthesis (EES). While not a replacement of standard evolutionary approaches, this is an expansion and enhancement of our toolkit. I offer brief examples from human evolution in support of these assertions.

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PACANINI, EMIL P., MOHAMED H. ABDULHADI, JOSE GARCIA i MAGNUS O. MAGNUSSON. "Recombinant Human Erythropoietin Correction of Anemia". ASAIO Transactions 35, nr 3 (lipiec 1989): 513–15. http://dx.doi.org/10.1097/00002216-198907000-00110.

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PACANINI, EMIL P., MOHAMED H. ABDULHADI, JOSE GARCIA i MAGNUS O. MAGNUSSON. "Recombinant Human Erythropoietin Correction of Anemia". ASAIO Transactions 35, nr 3 (lipiec 1989): 513–15. http://dx.doi.org/10.1097/00002480-198907000-00110.

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Cordone, L., A. Cupane, M. Leone, V. Militello i E. Vitrano. "Oxygenation of partially oxidized human hemoglobin". Biophysical Journal 63, nr 6 (grudzień 1992): 1678–80. http://dx.doi.org/10.1016/s0006-3495(92)81752-6.

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