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Development of Methods to Investigate Pulmonary Arterial Smooth Muscle Cells under Hypoxia
Luleå University of Technology, Department of Engineering Sciences and Mathematics, Fluid and Experimental Mechanics.
2019 (English)Licentiate thesis, comprehensive summary (Other academic)
Abstract [en]

Hypoxic pulmonary vasoconstriction (HPV) is a physiological response to localized alveolarhypoxia that is intrinsic to the pulmonary circulation. By hypoxia-induced contractionof pulmonary arterial smooth muscle cells (PASMCs), the pulmonary capillary bloodflow is redirected to alveolar areas of high oxygen partial pressure, thus maintaining theventilation-perfusion ratio. Although the principle of HPV was recognized decades agothe underlying pathway remains elusive. The patch clamp technique, imaging and Ramanspectroscopy are methods that can be used to investigate parts of the mechanisms. Toenable measurements at controlled oxygen concentrations a gas-tight microfluidic systemwas developed. In this thesis preparatory experiments to couple the gas-tight systemto a microscope that enabled simultaneous measurements with patch clamp, imagingand Raman spectroscopy are discussed. The patch clamp technique is to be used formeasurements on the dynamics of the ion-channels in the cellular membrane as well aschanges in membrane potential as a response to hypoxia. Imaging of PASMCs is requiredto successfully apply the patch clamp technique. Further, imaging will also reveal whetherthe mechanical response of HPV has been triggered, for this purpose image analysis forestimation of optical flow can be used. Raman spectroscopy enables measurements ofbiochemical changes in redox biomarkers, cytochrome c and NADH, of the mitochondrialelectron transport chain. This thesis shows that the gas-tight microfluidic system providesoptimal control of the oxygen content, in an experimantal setting where the patch clamptechnique can be applied. Raman measurements showed significantly larger variationsin spectra compared to an open fluidic system, which is the conventional approach.However, the results showed a need for improved Raman preprocessing. For this purposea Convolutional Neural Network (CNN) was trained using synthetic spectra that providedoptimal reconstruction of the Raman signal. Finally, simultaneous imaging and Ramanspectroscopy of red blood cells were performed in a home built microscope. The resultspave the way for measurements on PASMCs.

Place, publisher, year, edition, pages
Luleå: Luleå University of Technology , 2019.
Series
Licentiate thesis / Luleå University of Technology, ISSN 1402-1757
National Category
Medical Engineering Applied Mechanics
Research subject
Experimental Mechanics
Identifiers
URN: urn:nbn:se:ltu:diva-77140ISBN: 978-91-7790-513-4 (print)ISBN: 978-91-7790-514-1 (electronic)OAI: oai:DiVA.org:ltu-77140DiVA, id: diva2:1377308
Presentation
2020-01-24, E237, Luleå, 10:00 (English)
Opponent
Supervisors
Available from: 2019-12-11 Created: 2019-12-11 Last updated: 2020-01-13Bibliographically approved
List of papers
1. Single-Step Preprocessing of Raman Spectra using Convolutional Neural Networks
Open this publication in new window or tab >>Single-Step Preprocessing of Raman Spectra using Convolutional Neural Networks
2019 (English)In: Applied Spectroscopy, ISSN 0003-7028, E-ISSN 1943-3530, ISSN 0003-7028Article in journal (Refereed) Accepted
Abstract [en]

Preprocessing of Raman spectra is generally done in three separate steps: (1) cosmic ray removal, (2) signal smoothing, and(3) baseline subtraction. We show that a convolutional neural network (CNN) can be trained using simulated data tohandle all steps in one operation. First, synthetic spectra are created by randomly adding peaks, baseline, mixing of peaksand baseline with background noise, and cosmic rays. Second, a CNN is trained on synthetic spectra and known peaks.The results from preprocessing were generally of higher quality than what was achieved using a reference based onstandardized methods (second-difference, asymmetric least squares, cross-validation). From 105 simulated observations,91.4% predictions had smaller absolute error (RMSE), 90.3% had improved quality (SSIM), and 94.5% had reduced signal-tonoise(SNR) power. The CNN preprocessing generated reliable results on measured Raman spectra from polyethylene,paraffin and ethanol with background contamination from polystyrene. The result shows a promising proof of concept forthe automated preprocessing of Raman spectra.

Place, publisher, year, edition, pages
Sage Publications, 2019
Keywords
Raman spectroscopy, convolutional neural network, CNN, preprocessing, simulated data, chemometrics
National Category
Signal Processing
Research subject
Experimental Mechanics
Identifiers
urn:nbn:se:ltu:diva-77138 (URN)10.1177/0003702819888949 (DOI)
Funder
Swedish Foundation for Strategic Research , ITM17-0056Swedish Research Council, 2016-04220
Available from: 2019-12-11 Created: 2019-12-11 Last updated: 2019-12-11
2. Comparison between process simulation and deformation measured by defocused speckle photography
Open this publication in new window or tab >>Comparison between process simulation and deformation measured by defocused speckle photography
Show others...
2018 (English)In: Speckle 2018: VII International Conference On Speckle Metrology / [ed] M. Kujawińska ; L. R. Jaroszewicz, SPIE - International Society for Optical Engineering, 2018, Vol. 10834, article id 108341KConference paper, Published paper (Refereed)
Abstract [en]

Defocused laser speckle photography is used as a tool to measure the heat responses in a titanium component during laser heating. The evolution of the response is compared with a set of preprocessed Finite Element Simulations of the corresponding process with the aim to verify the simulation model and to find the simulation settings that best resemble the experimental results. The titanium component consists of a 300 x 100 mm2 substrate of thickness 3.2 mm on which a 200 x 30 x 11 mm3 ridge is built up using the laser metal deposition by wire process. The component is heated on the top of the ridge by a 300 W laser for 10 s and the deformation of the subtrate is followed throughout the heating-cooling cycle. The simulated deformation gradient is shown to resemble the measured response, and the magnitude of the response indicates that about 70 % of the laser power transferres into heat in the metal.

Place, publisher, year, edition, pages
SPIE - International Society for Optical Engineering, 2018
Series
Proceedings of SPIE - the International Society for Optical Engineering, ISSN 0277-786X ; 10834
Keywords
Laser speckle, deformation, in-process measurement
National Category
Applied Mechanics Other Mechanical Engineering
Research subject
Experimental Mechanics; Computer Aided Design
Identifiers
urn:nbn:se:ltu:diva-71895 (URN)10.1117/12.2319384 (DOI)000455052600052 ()2-s2.0-85058383509 (Scopus ID)9781510622975 (ISBN)
Conference
SPECKLE 2018: VII International Conference on Speckle Metrology, 9-12 September 2018, Janów Podlaski, Poland.
Funder
VINNOVA
Available from: 2018-12-05 Created: 2018-12-05 Last updated: 2019-12-11Bibliographically approved
3. Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions
Open this publication in new window or tab >>Development of a Gas-Tight Microfluidic System for Raman Sensing of Single Pulmonary Arterial Smooth Muscle Cells Under Normoxic/Hypoxic Conditions
Show others...
2018 (English)In: Sensors, ISSN 1424-8220, E-ISSN 1424-8220, Vol. 10, article id 3238Article in journal (Refereed) Published
Abstract [en]

Acute hypoxia changes the redox-state of pulmonary arterial smooth muscle cells (PASMCs). This might influence the activity of redox-sensitive voltage-gated K⁺-channels (Kv-channels) whose inhibition initiates hypoxic pulmonary vasoconstriction (HPV). However, the molecular mechanism of how hypoxia-or the subsequent change in the cellular redox-state-inhibits Kv-channels remains elusive. For this purpose, a new multifunctional gas-tight microfluidic system was developed enabling simultaneous single-cell Raman spectroscopic studies (to sense the redox-state under normoxic/hypoxic conditions) and patch-clamp experiments (to study the Kv-channel activity). The performance of the system was tested by optically recording the O₂-content and taking Raman spectra on murine PASMCs under normoxic/hypoxic conditions or in the presence of H₂O₂. Oxygen sensing showed that hypoxic levels in the gas-tight microfluidic system were achieved faster, more stable and significantly lower compared to a conventional open system (1.6 ± 0.2%, respectively 6.7 ± 0.7%, n = 6, p < 0.001). Raman spectra revealed that the redistribution of biomarkers (cytochromes, FeS, myoglobin and NADH) under hypoxic/normoxic conditions were improved in the gas-tight microfluidic system (p-values from 0.00% to 16.30%) compared to the open system (p-value from 0.01% to 98.42%). In conclusion, the new redox sensor holds promise for future experiments that may elucidate the role of Kv-channels during HPV.

Place, publisher, year, edition, pages
Basel, Switzerland: MDPI, 2018
Keywords
Raman spectroscopy, hypoxia, microfluidic system, redox reactions on single cell level
National Category
Applied Mechanics Fluid Mechanics and Acoustics
Research subject
Experimental Mechanics; Fluid Mechanics
Identifiers
urn:nbn:se:ltu:diva-71090 (URN)10.3390/s18103238 (DOI)000448661500066 ()30261634 (PubMedID)2-s2.0-85054841733 (Scopus ID)
Note

Validerad;2018;Nivå 2;2018-10-03 (svasva)

Available from: 2018-10-03 Created: 2018-10-03 Last updated: 2019-12-11Bibliographically approved

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