Basic and Clinical Understanding of Microcirculation

Microcirculation is key to providing enough nutrition and oxygen from head to toe. This is possible only through an extensive network of blood vessels spread around the body. Effect of microcirculation abnormalities stretch beyond one’s comprehension. The effects could be felt at any age, from the foetal life to the adulthood. The chapters present in this book describe how these abnormalities could lead to diseases such as atherosclerosis, thrombosis, diabetes, hypertension. Disorders of microcirculation could be related to the structural and/or functional damage to the inner lining of the blood vessels. Early identification of these disorders could benefit many ailments including cardiovascular and cerebrovascular diseases such as heart attack and stroke

Understanding human aging and the fundamental cell signaling link in age-related diseases: the middle-aging hypovascularity hypoxia hypothesis

Aging-related hypoxia, oxidative stress, and inflammation pathophysiology are closely associated with human age-related carcinogenesis and chronic diseases. However, the connection between hypoxia and hormonal cell signaling pathways is unclear, but such human age-related comorbid diseases do coincide with the middle-aging period of declining sex hormonal signaling. This scoping review evaluates the relevant interdisciplinary evidence to assess the systems biology of function, regulation, and homeostasis in order to discern and decipher the etiology of the connection between hypoxia and hormonal signaling in human age-related comorbid diseases. The hypothesis charts the accumulating evidence to support the development of a hypoxic milieu and oxidative stress-inflammation pathophysiology in middle-aged individuals, as well as the induction of amyloidosis, autophagy, and epithelial-to-mesenchymal transition in aging-related degeneration. Taken together, this new approach and strategy can provide the clarity of concepts and patterns to determine the causes of declining vascularity hemodynamics (blood flow) and physiological oxygenation perfusion (oxygen bioavailability) in relation to oxygen homeostasis and vascularity that cause hypoxia (hypovascularity hypoxia). The middle-aging hypovascularity hypoxia hypothesis could provide the mechanistic interface connecting the endocrine, nitric oxide, and oxygen homeostasis signaling that is closely linked to the progressive conditions of degenerative hypertrophy, atrophy, fibrosis, and neoplasm. An in-depth understanding of these intrinsic biological processes of the developing middle-aged hypoxia could provide potential new strategies for time-dependent therapies in maintaining healthspan for healthy lifestyle aging, medical cost savings, and health system sustainability

Microcirculation and inflammation in a numerical simulation approach

Inflammation is the response of the organism to eradicate the agent of lesion or infection in order to achieve hemostasis. This response requires the migration of specific leukocyte populations from the blood circulation towards the inflamed area. Leukocyte recruitment constitutes a complex cellular process by which leukocytes are first recruited to the endothelial vascular wall of post-capillary venules across which they further extravasate into the interstitial tissue. Recruitment is mediated via cell-cell interactions between the leukocyte and the endothelium and occurs through a multi-step cascade: tethering, rolling, slow rolling, arrest, crawling, adhesion and transmigration. However, whether or not the leukocytes adhere to the endothelium depends not only on the chemical forces generated by adhesion molecules on leukocytes and endothelial cells, but also on the physical forces that act on those cells. It has been suggested that fluid shear stress resulting from blood flow also regulates leukocyte activity which makes the fluid dynamic environment of the circulation to be considered an important aspect for leukocyte recruitment and migration during the inflammatory response. Most of the studies on the inflammatory response and in particular on leukocyte recruitment are based on animal models and involve, among others, the quantification of inflammatory mediators and cellular players, and/or the analysis of the leukocyte-endothelial cell interactions by intravital microscopy. However, the contribution of hemodynamics for leukocyte recruitment has been seldom addressed in those studies. This is mostly due to the fact that the study of hemodynamics in in vivo animal models is not straightforward and moreover, that several hemodynamic parameters cannot be experimentally determined due to technical constraints. In this work, we reasoned that these limitations could be circumvented by the development and use of numerical simulations to describe leukocyte recruitment. Many of the processes, which take place in living organisms, can be expressed as mathematical equations. This applies to leukocyte recruitment, for which scarce numerical models existed before the beginning of this work. Importantly, these mathematical simulations were performed without considering simultaneously all the players in the process, namely the vessel, the blood flow and the leukocytes. Moreover, most of these studies were two dimensional, assumed blood as a Newtonian fluid with constant viscosity and did not take into account in vivo experimental data. Taken this, our major goal with this work was to understand the contribution of hemodynamics to leukocyte recruitment in inflammation. For such purpose, we aimed here at developing numerical simulations that more adequately reproduced this process. For such, we set up animal models of inflammation to obtain the experimental data required for the development of those numerical simulations. Finally, we used these models to investigate the role of hemodynamics in leukocyte recruitment in inflammation. First, we considered the simpler case of a numerical simulation that assumed leukocytes to be rigid spheres and blood, a non-Newtonian fluid. For such, we initially developed an animal model of inflammation in Wistar rats using a lipopolysaccharide (LPS) as an inflammatory agent. Blood samples were collected for determination of TNF-α levels to ensure the triggering of the inflammatory process. Importantly, the number of rolling and adherent leukocytes in post-capillary venules was monitored using an intravital microscopy approach. As expected, our results showed that there is an increase in TNF-α concentrations after 15 minutes of LPS administration and a significant increase in the number of rolling and adherent leukocytes. The recorded intravital microcopy images, along with other recorded parameters, were then used, in collaboration with a group of mathematicians, to develop a numerical model capable of describing leukocyte recruitment in the microcirculation. To evaluate the contribution of hemodynamics, the localized velocity fields and shear stresses on the surface of leukocytes and near the vessel wall contact points have been computed in two discrete situations, namely as a single leukocyte or when a cluster of them are recruited towards the vessel wall. In the first situation, our numerical results showed the presence of one region of maximum shear stress on the surface of the leuko- cyte close to the endothelial wall and of two regions of minimum shear stress on the op- posite side of the cell. The different areas of shear stress observed in the surface of the leukocyte may be important in directing it towards the endothelial wall during an inflammatory response. The identification of a region of maximum shear stress is consistent with the molecular mechanisms that govern leukocyte rolling because it may actually cor- respond to the area that supports the interaction with the endothelium. On the other hand, the relatively lower shear stress regions may correlate with leukocyte surface areas where binding to the endothelium is not occurring at the moment, thus enabling the roll- ing of the cell along the endothelium. It was also observed that the shear stress at the endothelium gets higher as a cluster of leukocytes moves in the main stream. This sug- gests that the presence of a cluster of leukocytes may potentiate leukocyte rolling, as the increase in the shear stress promoted by the recruited leukocytes may support the migra- tion and recruitment of additional cells. Despite closely simulating leukocyte recruitment, our initial numerical simulation consid- ered the simple case of leukocytes as rigid spheres. However, while circulating leukocytes maintain an approximately spherical shape, rolling leukocytes are known to deform. In order to account for the leukocyte deformability changes that occur during its recruit- ment in inflammation, we needed to assess the deformability profile of the leukocytes under flow and therefore, to “directly” observe them regardless of the other blood cells. For such, intravital microscopy was performed in the mouse cremaster of a transgenic mice strain (Lys-EGFP-ki) in which fluorescent neutrophils can be individually tracked. By using PAF as an inflammatory agent, the analysis of the leukocyte-endothelial cell interac- tions showed a continuous increase in the number of rolling and adherent neutrophils up to 4 hours after the introduction of the inflammatory stimuli, thus confirming the devel- opment of an inflammatory response. As the properties of the red blood cells modulate blood flow properties, erythrocyte deformability was also addressed in this model. A con- tinuous decrease of this parameter was observed throughout time. The decrease in the erythrocyte deformability will most probably lead to an increase in the blood viscosity and to the decrease of the blood flow velocity. These conditions should facilitate the mi- gration of leukocytes from the mainstream to the endothelial wall and promote leukocyte slow rolling and adhesion during the inflammatory response. Importantly, in the intravital microcopy images obtained with this latter model, we clearly observed the deformation of neutrophils along the endothelial wall during rolling, as well as the formation of tethers. As such, in these images, leukocyte trajectories were tracked and their velocities and diameters were measured and further applied to the numerical simulations. Using a recent validated mathematical model describing the coupled defor- mation-flow of an individual leukocyte and the respective experimental results, numerical simulations of the recruitment of an individual leukocyte and of two leukocytes under different velocities were performed, considering a constant blood viscosity. The mathe- matical models obtained showed that under conditions of increased velocity the cell movement is accelerated along the endothelial layer, favouring the dissociation of leuko- cyte-endothelium interactions at designated attraction points. These observations lead us to propose that, in order to attain an efficient inflammatory response, the blood flow ve- locity needs so as to decrease to facilitate slow rolling and subsequent adhesion. These results are corroborated by the decrease in the erythrocyte deformability observed in our animal model, which will ultimately have an impact on the blood flow velocity. Our results further showed that in the vicinity of an adherent leukocyte there is an early slight decel- eration of the rolling leukocyte when compared with the case of an individual leukocyte. As such, these observations strongly suggest that the presence of an adherent cell in the vicinity should decrease the velocity of another leukocyte that is being recruited, thus promoting its slow rolling, and contributing to its capture by the endothelial cells. Altogether, our experimental data and numerical simulations support our working hy- pothesis that the hemodynamic properties of the flow and of the cells in circulation should play an essential role in the margination and rolling of the leukocytes to the endo- thelial wall, which in turn will impact the success of the inflammatory response. In partic- ular, our results strongly suggest that changes in hemodynamic conditions, such as de- creased flow velocities and the increase of the shear stress, will contribute to target leu- kocytes to the endothelial wall. Given our results, we propose that any change in the he- modynamic properties will certainly influence the outcome of the inflammatory response. As such, the adherence of the leukocytes to the endothelium should depend not only on the relative magnitude of the chemical forces generated by the interaction of adhesion molecules between leukocytes and endothelial cells, but also on the physical forces that act on the leukocytes. In this respect, our results suggest that alterations in the blood flow, for example in the flow velocity, will occur during an inflammatory process, thus potentiating the recruitment of more leukocytes towards the inflamed area and contrib- uting to a successful inflammatory response. Overall, the numerical simulations allowed us to better understand the contribution of the hemodynamic properties of the flow to the progression of an inflammatory response and to deepen our knowledge on leukocyte recruitment in inflammation. Importantly, our work provided numerical tools that can be used for the subsequent study and modulation of the hemodynamic parameters involved in an inflammatory response. In particular, these numerical simulations will surely enable us, in the near future, to determine or es- timate a large set of parameters which are unlikely to be recoverable by in vivo experi- ments. Moreover, our methods will allow us to analyze how the parameters evolve over time. Altogether our results further reinforce the notion that the improvement and de- velopment of animal models and numerical tools will certainly provide the medical and biological community with useful tools to study leukocyte recruitment in inflammation. By closely reproducing the microcirculation and the inflammatory process, these tools will be critical for a better comprehension of the inflammatory process and of the mecha- nisms underlying a multitude of inflammatory pathological conditions

Availability of metal ions and ZnO nanoparticles in aqueous media

La presència de determinats metalls, ja sigui en forma del metall lliure, formant complexos amb lligands o en forma de nanopartícules metàl•liques té efectes negatius en el medi ambient. Per aquest motiu, l’estudi de la disponibilitat de metalls és important per determinar el seu impacte mediambiental. En aquest treball s'ha estudiat un sistema i una tècnica per a la mesura de la disponibilitat. El sistema té la particularitat de contenir, en dispersió, nanopartícules d’òxids metàl•lics. Una primera part de l’estudi s’enfoca a la caracterització físic-química de dispersions sintètiques de nanopartícules. Aquesta part estudia l’efecte que tenen les condicions del medi (pH, salinitat, contingut de matèria orgànica, etc.) en el comportament de les nanopartícules. S'han determinat processos com són l’agregació, solubilització i sedimentació, en funció de les condicions del medi. Tots aquests processos juguen un paper molt important en el destí de les nanopartícules en el medi ambient i en la seva toxicitat. La segona part d’aquesta tesi es centra en l’aplicació de la tècnica analítica DGT (Diffusive Gradient in Thin films) per la mesura de fluxos de metall disponibles en solucions aquoses. En el present treball, s’ha determinat la solubilitat de nanopartícules en dispersions aquoses. Per altra banda s'ha estudiat la tècnica DGT des d’un punt de vista físic-químic. Les contribucions dels fluxos de complexos s’avaluen amb un paràmetre que té en compte les constants de dissociació dels complexos així com la seva difusió. S'han estudiat les acumulacions de metalls que en solució tenen com especies dominants complexos carregats elèctricament. Si aquesta càrrega és negativa, l’acumulació disminueix en baixar la força iònica i a l’inrevés en el cas contrari. Aquest fenomen s’explica per la repulsió electrostàtica entre la càrrega negativa del disc de resina i el complex quan aquest és parcialment làbil. En conclusió, la resina sembla jugar un paper més actiu del que es pensava en l’acumulació de metalls en DGT.La presencia de determinados metales, ya sea en forma del metal libre, formando complejos con ligandos o en forma de nanopartículas metálicas tiene efectos negativos en el medio ambiente. Por este motivo, el estudio de la disponibilidad de metales es importante para determinar su impacto medioambiental. En este trabajo se ha estudiado un sistema y una técnica para la medida de la disponibilidad. El sistema tiene la particularidad de contener, en dispersión, nanopartículas de óxidos metálicos. Una primera parte del estudio se enfoca a la caracterización físico-química de dispersiones sintéticas de nanopartículas. Esta parte estudia el efecto que tienen las condiciones del medio (pH, salinidad, contenido de materia orgánica, etc) en el comportamiento de las nanopartículas. Se han determinado procesos como son la agregación, solubilidad y sedimentación, en función de las condiciones del medio. Todos estos procesos juegan un papel muy importante en el destino de las nanopartículas en el medio ambiente y en su toxicidad. La segunda parte de esta tesis se centra en la aplicación de la técnica analítica DGT (Diffusive Gradiente in Thin films) para la medida de flujos de metal disponibles en soluciones acuosas. En el presente trabajo, se ha determinado la solubilidad de nanopartículas en dispersiones acuosas. Por otra parte se ha estudiado la técnica DGT desde un punto de vista físico-químico. Las contribuciones de los flujos de complejos se evalúan con un parámetro que tiene en cuenta las constantes de disociación de los complejos así como su difusión. Se han estudiado las acumulaciones de metales que en solución tienen como especies dominantes complejos cargados eléctricamente. Si esta carga es negativa, la acumulación disminuye al bajar la fuerza iónica y viceversa en el caso contrario. Este fenómeno se explica por la repulsión electrostática entre la carga negativa del disco de resina y el complejo cuando éste es parcialmente lábil. En conclusión, la resina parece jugar un papel más activo de lo que se pensaba en la acumulación de metales en DGT.The presence of certain metals, either in the form of free metal, forming complexes with ligands or as metal nanoparticles has negative effects on the environment. For this reason, the study of the availability of metals is important in determining its environmental impact. In this work a system and a technique for measuring availability have been studied. The system contains, in dispersion, metal oxide nanoparticles. A first part of the study focuses on the physical and chemical characterization of synthetic dispersions of nanoparticles. This part examines the effect of environmental conditions (pH, salinity, organic matter content, etc.) in the behavior of nanoparticles. Processes such as aggregation, solubilisation and sedimentation, are determined depending on environmental conditions. All these processes play an important role in the fate of nanoparticles in the environment and their toxicity. The second part of this thesis focuses on the application of the analytical technique DGT (Diffusive Gradient in Thin films) for measuring the flow of available metals in aqueous solutions. In the present study, the solubility of nanoparticles in aqueous dispersions has been determined. Moreover the DGT technique has been studied from a physical-chemical point of view. The contribution of the flux of complexes has been evaluated with a parameter, the lability degree, which takes into account the dissociation constants of the complexes and their diffusion. The accumulations of metals which in solution are present as dominant metal complexes electrically charged have been studied. If the charge is negative, the accumulation decreases with the decrease of the ionic strength and vice versa in the opposite case. This phenomenon is explained by the electrostatic repulsion between the negatively charged resin disk and the complex when it is partially labile. In conclusion, the resin seems to play a more active role than previously thought in the accumulation of metals in DGT

Resolving the liver sinusoidal endothelial phenotype in health and disease

The burden of liver disease is continuously increasing globally, and this emphasises the need for the development of therapeutics. In order for this to be achieved, potential cellular and molecular targets need to be identified. Liver sinusoidal endothelial cells (LSECs) play a key role in maintaining liver homeostasis and their dysfunction drives liver disease pathophysiology and this role needs to be further elucidated. In order to identify phenotypic differences in LSECs in health and disease, a combination of analytical techniques such as immunohistochemistry and qPCR was applied on human tissue specimens. To confirm whether these changes are recapitulated in vitro, I isolated LSECs from human healthy and cirrhotic tissue specimens for the establishment of culture model of human LSECs. Validation of functional and phenotypic characteristics of LSECs in vitro was carried out using immunocytochemistry and qPCR. Furthermore, the development and optimisation of a super-resolution imaging protocol for the visualisation of LSEC fenestrations was performed. Altered expression and downregulation of scavenger receptors in LSECs was identified in diseased human tissue specimens compared to healthy specimens and this confirmed capillarisation of sinusoidal endothelial cells in liver disease. Expression of scavenger receptors and key regulatory molecules was maintained in LSECs in vitro. The phenotypic changes in LSECs identified in liver tissue specimens were partially recapitulated in LSECs in vitro. The application of pharmaceutical molecules for the enhancement of nitric oxide (NO) signalling in LSECs revealed an altered genotype in healthy and cirrhotic LSECs. Finally, fenestrations were visualised on the LSEC membrane using the developed super-resolution imaging protocol and improvement in LSEC porosity following the application of sildenafil citrate. Hence these findings emphasise the relevance of appropriate culture models and imaging approaches to study phenotypic changes in LSECs in relation to disease and highlight the therapeutic potential of sildenafil citrate in improving LSECs porosity

Benthic diatom monitoring and assessment of freshwater environments: standard methods and future challenges

Fil: Soizic, Morin. National Research Institute of Science and Technology for Environment and Agriculture; FranceFil: Gómez, Nora. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología Dr. Raúl A. Ringuelet; ArgentinaFil: Tornés, Elisabet. University of Girona. Institute of Aquatic Ecology; SpainFil: Licursi, Magdalena. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología Dr. Raúl A. Ringuelet; ArgentinaFil: Rosebery, Juliette. Aquatic Ecosystems and Global Changes Research Unit; Franc

Benthic diatom monitoring and assessment of freshwater environments: standard methods and future challenges

Fil: Soizic, Morin. National Research Institute of Science and Technology for Environment and Agriculture; FranceFil: Gómez, Nora. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología Dr. Raúl A. Ringuelet; ArgentinaFil: Tornés, Elisabet. University of Girona. Institute of Aquatic Ecology; SpainFil: Licursi, Magdalena. Universidad Nacional de La Plata. Facultad de Ciencias Naturales y Museo. Instituto de Limnología Dr. Raúl A. Ringuelet; ArgentinaFil: Rosebery, Juliette. Aquatic Ecosystems and Global Changes Research Unit; Franc

Odyssée au fil des interfaces: de la physico-chimie des macromolécules à l'enveloppe bactérienne, plate-forme interactive du micro-organisme avec son micro-environnement

This find is registered at Portable Antiquities of the Netherlands with number PAN-0001909

The environmental toxicology of zinc oxide nanoparticles to the oligochaete

This thesis investigated the potential toxicity of zinc oxide nanoparticles (NPs) and bulk particles (both with and without organic matter (HA)) to the Californian Blackworm, Lumbriculus variegatus. The NPs and bulk particles in this thesis were characterised using numerous techniques. ZnO NPs were found to be 91 (13364) nm (median (interquartile range)) and ZnO bulk particles were found to be 237 (322165) nm (median (interquartile range)) by TEM. In the acute behavioural study (96 hour), ZnO NPs had a dose-dependent toxic effect on the behaviour of the worms up to 10mg/L whereas the bulk had no significant effect. This result, however, was mitigated by the addition of 5mg/L HA in the NP study whereas a similar addition enhanced the toxicity of the bulk particles at 5mg/L ZnO. In the chronic study (28 days), ZnO NPs and bulk particles were found to have a dose-dependent significant effect on the behaviour of the worms after 28 days, with NPs causing a significantly greater negative response than bulk particles at 12.5, 25 and 50mg/L ZnO. HA had no effect on the toxicity of either particle type in the chronic study. Acute (96 hour) oxidative stress in L. variegatus in response to ZnO NP and bulk particle (with and without 5mg/L HA) exposure was evaluated by examining the changes in glutathione (GSH) content of cells, since NPs are expected to have potential for toxicity via mechanisms such as oxidative stress. Neither ZnO NPs nor bulk particles (with and without 5mg/L HA) were found to induce significant changes in the GSH content of L. variegatus cells after 96 hours of exposure. The uptake, accumulation and depuration of ZnO NPs and bulk particles in L. variegatus over a 48 hour period of uptake and a 48 hour period of depuration were also investigated. This study found a high level of variability and it was concluded that the protocol employed was not suitable for investigating the uptake, bioaccumulation and depuration of ZnO NPs and bulk particles. Finally, histological techniques and a number of fixatives were evaluated for use with L. variegatus. Bouin’s solution was found the most suitable fixative for use with these worms, with no histological damage observed in the morphology of the worms after a 96 hour exposure to ZnO NPs and bulk particles (with and without 5mg/L HA). When considering the toxicology results from all experiments within this thesis it is concluded that ZnO NPs can cause both acute and chronic toxicity in terms of behavioural response, but do not cause acute oxidative stress in L. variegatus