Cardiovascular diseases are a major cause of mortality and morbidity in the developed countries. Anti-platelet therapy is a cornerstone treatment for patients with cardiovascular diseases. Patients are routinely managed with a combination therapy consisting of aspirin and clopidogrel. Aspirin inhibits cyclooxygenase 1 (COX 1) a crucial intermediate enzyme involved in thromboxane biosynthesis. Clopidogrel on the other hand antagonizes ADP receptor P2Y12. ADP is a weak platelet agonist stored in platelet dense granules and is released upon platelet activation. ADP activates platelets through two purinergic receptors namely P2Y1 and P2Y12 these receptors couple to Gq and Gi class of G-proteins, respectively. P2Y1 causes calcium mobilization through activation of PLC-β. P2Y12 inhibits adenylyl cyclase, causes activation of Rap1B and Akt. Signaling from both the receptors is required for complete integrin activation, thromboxane generation and Erk activation. Previous studies have shown that P2Y12 potentiates fibrinogen receptor activation, secretion, thrombi stabilization, thrombin generation, platelet leukocyte aggregation formation. ThromboxaneA2 (TXA2) is a potent platelet agonist generated through arachidonic acid metabolism in platelets. TXA2 thus, generated after platelet activation acts as a positive feedback mediator along with ADP. Under physiological conditions, platelet activation leads to thrombin generation through coagulation cascades. Generated thrombin activates PAR receptors and ADP is released from dense granules, which further potentiates thromboxane generation downstream of PARs. Current anti-platelet therapy regimens often include P2Y12 antagonists and aspirin in management of patients with acute coronary syndrome (ACS) and in those undergoing percutaneous coronary intervention (PCI) with stent implantation. However, there still exists a need for improved treatment strategies as not all patients benefit from this dual combination therapy. Reasons include, poor responders either to P2Y12 antagonists or to aspirin, or if aspirin is contraindicated in these patient populations. In the current study we evaluated the role of P2Y12 in thromboxane generation under physiological conditions. We studied serum thromboxane generation in a model system wherein P2Y12 was antagonized or deficient. Using pharmacological approaches we show that dosing mice with 30mg/Kg/body weight clopidogrel or 3mg/Kg/body weight prasugrel decreased serum thromboxane levels when compared to the control mice. Pre-treatment of human blood ex vivo with active metabolites of clopidogrel (R361015) or prasugrel (R138727) also led to reduction in thromboxane levels. We also evaluated serum thromboxane levels in P2Y receptor null mice, serum thromboxane levels in P2Y1 null mice were similar to those in wild type littermates, and were inhibited in P2Y12 null mice. Furthermore, serum thromboxane levels in P2Y12 deficient patients, previously described in France and Japan, were also evaluated and these patients had lower serum thromboxane levels compared to normal controls. In a pilot study, serum thromboxane levels were radically reduced in healthy human volunteers upon dosing with clopidogrel, compared to the levels before dosing. In conclusion, P2Y12 antagonism alone can decrease physiological thromboxane levels. Thus P2Y12 regulates physiological thromboxane levels. Further it is known that ADP-induced thromboxane generation is integrin-dependent. However it is not clear if other potent platelet agonists like thrombin require outside-in signaling for thromboxane generation. Our results show that thrombin-induced thromboxane generation was independent of integrins i.e. when platelets were stimulated with PAR agonists in presence of fibrinogen receptor antagonist thromboxane generation was not affected. Since PAR agonists, unlike ADP, activate G12/13 signaling pathways. Hence, we hypothesized that these pathways might play a role in TXA2 generation. Our results show, that inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by costimulation of G12/13 pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G12/13 pathways. Next, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. Our results showed that c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. We observed differential activation of FAK downstream of integrins and G12/13 pathways. ADP-induced activation of FAK was integrindependent and SFK-independent. On the other hand selective activation of G12/13 pathway lead to FAK activation, in SFK and Rho dependent manner. We also evaluated specificity of new FAK inhibitor TAE-226 to understand the role of FAK in TXA2 generation. Our results showed that TAE-226 exhibited non-specific effects at higher concentrations. Furthermore, in comparison to WT mice, FAK null mice did not show any difference in TXA2 generation. Therefore, we concluded that differential activation of FAK occurs downstream of Integrins and G12/13 pathways. However, the common effector molecule downstream of integrins and G12/ 13 pathways contributing to TXA2 generation in platelets remains elusive. / Molecular and Cellular Physiology

The purpose of this research was to study vitamin A status in grazing horses throughout the year and to evaluate the effectiveness of vitamin A and β-carotene as supplements. Vitamin A status was assessed by serum retinol concentrations (SR) and the relative dose response (RDR) which was adapted for use in the horse. The horses (45 Thoroughbred mares) were divided into three diet groups: pasture and hay only (PH); pasture, hay and vitamin A-free concentrate (PHC); and hay and vitamin A-free concentrate (HC). The mares, as well as their foals, were assessed for vitamin A status during the summer, fall and winter. After eight months, each diet group was subdivided and supplemented with either: retinyl palmitate at two times the recommended level (A), the equivalent in water dispersible β-carotene (B), or a placebo (C). Supplementation continued for 20 months during which the vitamin A status was assessed every 60 days in the mares, and at birth in the neonates. During both the depletion and the repletion phase the mares were kept on a regular breeding schedule and the reproductive rates were determined as well as the general health of the mares and their offspring. The RDR proved more sensitive at detecting changes in vitamin A status than SR, and a combination of both was used. A measurable decline in vitamin A stores was seen in the HC group within 2 months, and in PH and PHC groups during the winter. The HC group remained lower in vitamin A status throughout the study. A seasonal fluctuation of vitamin A status was observed regardless of supplementation. Supplementation with retinyl palmitate improved vitamin A status in all three diet groups, however, supplementation with β-carotene did not. Both neonates and young growing horses were lower in vitamin A status than the adult. A respiratory infection observed in the weanlings affected vitamin A status as well. Supplementation of the dam had no effect on neonatal vitamin A status. Deleterious effects on reproductive rates and health were also observed with vitamin A depletion. Supplementation of β-carotene had a negative effect on reproductive rates in this study. / Ph. D.

The emergence of microneedle (MN) technologies offers a route for a pain free, straightforward and efficient way of transdermal drug delivery, but technological barriers still exist which pose significant challenges for manufacture of MN systems with high volume outputs at low cost. The main aim of this research was to develop new ways for MN manufacture primarily using micro-injection moulding processes with high performance engineering thermoplastics. During the moulding process these polymeric melts will be subjected to extreme stress and temperature gradients and detailed material characterisation combined with in-line monitoring is desirable to optimise the moulding parameters and will help in achieving sharp microneedles with acceptable quality. Hence high shear rheology of these selected materials was performed at wall shear rates carried out in excess of 107 s-1 over a range of temperatures to predict the flow behaviour of polymer melts at such high shear strain rates. This information was fed into injection moulding simulation software tools (Moldflow) to assist the MN production process design. The optimal design was then used to produce a full 3D solid model of the injection mould and mould insert. Furthermore various design of experiments were conducted considering input parameters such as injection pressure, injection speed, melt temperature, filling time and mould cavity temperature. Response variables including product quality and data acquired from the cavity pressure and temperature transducers were used to optimise the manufacturing process. The moulded MNs were geometrically assessed using a range of characterisation techniques such as atomic force microscopy, confocal microscopy and scanning electron microscopy. An attempt to make hollow MNs was performed and encountered many challenges like partial cavity filling and part ejection during processing. Studies were carried out to understand the problem and identified the major problem was in tool design and improvements to the moulding tool design were recommended. Plasma treatment and mechanical abrasion were employed to increase the surface energy of the moulded polymer surfaces with the aim of enhancing protein adsorption. Sample surface structures before and after treatment were studied using AFM and surface energies have been obtained using contact angle measurement and calculated using Owens-Wendt theory. Adsorption performance of bovine serum albumin and release kinetics for each sample set was assessed using a Franz diffusion cell. Results indicate that plasma treatment significantly increases the surface energy and roughness resulting in better adsorption and release of BSA. To assist design-optimisation and to assess performance, a greater understanding of MN penetration behaviour is required. Contact stiffness, failure strength and creep behaviour were measured during compression tests of MN against a steel surface, and in-vitro penetration of MNs into porcine skin. The MN penetration process into porcine skin was imaged using optical coherence tomography. Finally, a finite element model of skin was established to understand the effect of tip geometry on penetration. The output of findings from this research will provide proof of concept level development and understanding of mechanisms of MN penetration and failure, facilitating design improvements for micro-injection moulded polymeric MNs.

High body weight seems protective of bone mass, specifically bone mineral content (BMC) and bone mineral density (BMD), thereby reducing the risk of osteoporosis. Cognitive eating restraint (CER), diet composition, and the satiety hormone, leptin, produced by adipocytes, are associated with body mass and may also influence bone mass. Few studies have examined these relationships. To investigate the relationship between leptin and CER score, 36 premenopausal, healthy weight women, as defined by body mass index (BMI) of 18-25 kg/m², aged 18-25 years were studied. Women were categorized by baseline Eating Inventory questionnaire scores into either the high CER group (score > 9, n = 20) or low CER group (score < 9, n = 16). Serum leptin concentration was significantly lower in the low CER group versus high CER group at baseline. A positive relationship between serum leptin concentration and body fat mass and body fat % in normal weight women despite differences in CER scores was observed. In a separate study, overweight and obese women, (BMI > 25 to < 43 kg/m²), aged 32-45 years, were randomly assigned to either a low-carbohydrate, high-protein (LCHP) or low-fat, high-carbohydrate (LFHC) diet for 12 weeks. Serum leptin concentration was significantly greater in the LCHP versus the LFHC diet group at 12 weeks (p < 0.05). Over time, significant decreases in serum leptin concentration, BMI, body weight, total lean mass, total fat mass, and body fat % were observed in both diet groups. Serum leptin concentration was positively associated with body weight, fat mass, and body fat % regardless of diet consumed. Both studies are novel in their respective populations and show no direct link between leptin and bone mass when considered in the context of CER or diet composition. / Master of Science

Laktatdehydrogenas (LD) är en vanlig biomarkör som används för att uppskatta allmän vävnadsskada och celldöd i kroppen. Det mäts i blodserum och analyseras, liksom de flesta andra blodprover, så snart som möjligt efter korrekt förberedelse. Det kan dock uppstå förseningar i analysen som leder till att analyser måste senareläggas vilket i sin tur kan leda till nedbrytning av vissa molekyler och kemiska strukturer i prover. Studien syftade till att undersöka om, och i så fall i vilken omfattning, nedbrytning på grund av förvaring i rumstemperatur (22 °C) respektive kyla (4 °C) som påverkar de kvantifierbara resultaten av LD inom 30 timmar efter att proverna tagits. För att uppnå detta togs blodprover (n = 30) från inneliggande patienter där serum alikvoterades till två rör där ena röret förvarades i rumstemperatur och andra i kylskåp varpå upprepade mätningar av LD utfördes under totalt 26 timmar. Resultaten visade att förvaringstemperatur och tid inte hade någon statistiskt signifikant påverkan på värdet av LD (p &gt; 0,05) och att LD därmed kan mätas oavsett förvaring i kyla eller rumstemperatur inom 29 timmar efter provtagningen.

Yes / Plasma technology has been widely used to increase the surface energy of the polymer surfaces for many industrial applications; in particular to increase in wettability. The present work was carried out to investigate how surface modification using plasma treatment modifies the surface energy of micro-injection moulded microneedles and its influence on drug delivery. Microneedles of polyether ether ketone and polycarbonate and have been manufactured using micro-injection moulding and samples from each production batch have been subsequently subjected to a range of plasma treatment. These samples were coated with bovine serum albumin to study the protein adsorption on these treated polymer surfaces. Sample surfaces structures, before and after treatment, were studied using atomic force microscope and surface energies have been obtained using contact angle measurement and calculated using the Owens-Wendt theory. Adsorption performance of bovine serum albumin and release kinetics for each sample set was assessed using a Franz diffusion cell. Results indicate that plasma treatment significantly increases the surface energy and roughness of the microneedles resulting in better adsorption and release of BSA.

This study investigates the efficacy of a newly developed humanized serum-free media (SFM) that replaces Fetal Bovine Serum (FBS), in culturing of common in vitro models (Caco-2 and 3D Primary Human Hepatocytes (PHH)). The research’s focus aligns with the 3R principles (replacement, reduction, refinement) and may potentially lead to enhancing the human relevance of the in vitro models and ultimately contribute to more accurate and successful drug development in the future. This study is divided into two subprojects, Caco-2 and 3D PHH, respectively. For Caco-2, we evaluated cell morphology, proliferation and monolayer formation by assessing the permeability of marker compounds. For PHH, we investigated spheroid formation and viability using an ATP measurement assay, lipid concentration using an Adipored Assay and CYP activity of the 3D PHH. Finally, global proteomics analysis was performed for both Caco-2 cells and 3D PHH grown in SFM and in conventional medium (CM). Our findings show that Caco-2 cells could proliferate well enough in the SFMCaco-2 without any change in their cell morphology, whereas their monolayer tightness was found to be similar in SFMCaco-2 as compared to CMCaco-2. Furthermore, from the proteomics analysis there was no major difference detected between cells in SFMCaco-2 and in CMCaco-2, however several of the relevant drug transporters of this model were found in higher levels in Caco-2 cultured in SFMCaco-2. For 3D PHH, our findings indicate that spheroids could be perfectly formed in both SFM3D PHH and in CM3D PHH, having relatable viability and lipid concentration, especially after two week of culturing period. Finally, the proteomics analysis revealed no significant difference between the proteome of the spheroids when cultured in SFM3D PHH or in CM3D PHH. In conclusion, these results suggest that the SFM provides a viable and sometimes superior alternative to CM for both Caco-2 and 3D PHH, promoting the 3R principles and potentially offering a more human-relevant model for in vitro studies.

This work is concerned with an attempt to clarify the role of cortisone in both the immune complement response and the progression of mouse leukemic tumor.

<p>U ovom radu je ispitivan uticaj Antitoksičnognutritiva(ATN), preparata na bazi prirodnih aluminosilikata,na biohemijske, fiziolo&scaron;ke, tehnolo&scaron;ke i proizvodne parametre uzgoja pilića. ATN je sme&scaron;a zeolita klinoptilolita, gline monmorilonita i male količine aktivnog uglja. Dodatak ovog preparata u hranivo za piliće u količini od 5 g/kg nije izazvao promene u normalnoj biohemijskoj i fiziolo&scaron;koj homeostazi životinja. Hematolo&scaron;ki parametri, koncentracija metabolita, elektrolita i aktivnost enzima seruma i jetre je bila u granicama referentnih vrednosti. ATN ne utiče na prirast životinja, ni na konverziju hrane, ali dovodi do povećanja relativnih masa pojedinih organa digestivnog trakta.</p><p>Uočava se da dodatkom ATN-a u hranivo dolazi do smanjenja količine masti a povećanja količine proteina u belom mesu. ATN takođe povećava i sadržaj pepela u<br />belom i crvenom mesu.</p><p>Akutni ili hronični tronedeljni oralni unos pojedinih toksikanata (mikotoksina aflatoksina B1i ohratoksina A, herbicida parakvata, jona olova ili toksina<br />cijanobakterija-mikrocistisa) dovodi do poremećaja normalne biohemijske i fiziolo&scaron;ke homeostaze pojedinih organa i tkiva pilića, &scaron;to je utvrđeno na osnovu rezultata hematolo&scaron;kih i biohemijskih analiza, određivanja enzima antioksidativne za&scaron;tite i lipidne&nbsp;<br />peroksidacije, kao i odredjivanja parametara uzgoja i težine organa. ATN, dodat u hranu u količini od 5 g/kg, mogao da bude dobar protektivni agens za delovanje aflatoksina, parakvata, jona te&scaron;kih metala i mikrocistisa, ali ne i ohratoksina.</p> / <p>This study investigated the effects of dietary supplementation with hydrated aluminosilicate (Antitoxic Nutrient-ATN), based on zeolitic ore (clinoptilolite), clay bentonite (montmorillonite),and small amounts of activated charcoal, on&nbsp; performance, hematological, serum, and liver biochemical parameters, as well as organ weights and meat quality in broiler chickens. The dietary addition of ATN has&nbsp; no adverse effects on serum and liver biochemical parameters and does not affect the normal physiological homeostasis of animals. However, the results of this study demonstrate that supplementation with 5 g/kg of ATN influenced organ weights, and chemical composition of broiler chicken meat.</p><p>This study also evaluated the effectiveness of ATN to protect broilers from adverse effects of five different toxic substances (mycotoxins aflatoxin B1, and ochratoxin A, herbicide paraquat, heavy metal ions supplied as lead-acetate, and microcystis, toxin of cyanobacteria). Toxic substances induced oxidative stress and disturb normal biochemical and physiological homeostasis of different tissues and organs in poultry. The results from this study demonstrate that the biochemical variables of serum, liver, kidney, lung and other organs were negatively affected by all five toxic substances. The additionof 5 g/kg of ATN was protective against aflatoxin B1, lead-acetate, paraquat and microcystis, but not against ochratoxin A.</p>

Hyperfosforylering av biomarkörproteinet Tau förekommer i flera neurodegenerativa sjukdomar som kallas Taupathies. Proteinets huvudfunktion i människokroppen är att modulera flexibilitet och stabilitet för axonal-mikrotubulin. I Taupathies utlöser hyperfosforyleringen av Tau instabilitet och neurodegenerationen. I dagens läge kan hyperfosforylering av treonin 217 (P217) endast mätas i hjärnan. I den här studien undersöks hyperfosforyleringen av treonin 217 (P217). I syfte att se om nivåerna av P217 är mätbara i cerebrospinalvätska (CSV) och i blodet. Samt för att evaluera hur nivåer av P217 förändras i olika Taupathies, genom att testa hjärnprover från friska kontroller och olika Taupathies. Studien görs för att öka kunskapen om effekten av hyperfosforylering av treonin 217 i Taupathies och för att bidra med en ny provtagningsmetod för P217. Simoa HD-1 Analyzer var instrumentet som användes för analyserna av P217. Det är ett instrument som kan upptäcka onormala nivåer av biomarkörer genom kvantifiering, med hjälp av antikroppar och ett enzym. Enzymet kallas Streptavidin β-galaktosidas och omvandlar en befintlig P217-molekyl i proven till en fluorescerande produkt. Genom Simoa HD-1 Analyzer utvecklades en ultrasensitiv analys med antikropparna P217 och Tau 12, som kunde upptäcka mycket låga nivåer av P217 i hjärnan, CSF och i blod. Förändring av P217-nivåer hittades även i olika Taupathies. De Taupathies med de högsta nivåerna av P217 var Progressiv supranukleär pares, Corticobasal degeneration och Globular glial Taupathies. / Hyperphosphorylation of the biomarker protein Tau occurs in many neurodegenerative diseases called Taupathies. The proteins main function in the human body is to modulate flexibility and stability for axonal microtubules. In Taupathies the hyperphosphorylation of the Tau triggers instability and neurodegeneration. Nowdays hyperphoshorylation on threonine 217 (P217) can only be measured in the brain. In this study the hyperphoshorylation on the phosphorylation site of threonine 217 (P217) is examined. In aim to see if levels of P217 is measurable in cerebrospinal fluid (CSF) and in blood. As well to evaluate how P217 variate in different Taupathies, through the use of brain samples from healthy controls and different Taupathies. The study is made for the purpose of enhancing the pure knowledge about the effect of hyperphosphorylation on threonine 217 in Taupathies and to contribute with a new sampling method for P217. Simoa HD-1 Analyzer was the key instrument of the analyses of P217. It’s an instrument which can detect abnormal levels of biomarkers through quantification, with help of antibodies and an enzyme. The enzyme is called Streptavidin β-galactosidase and converts an existing P217 molecule in the samples to a fluoresce product. Through the use of Simoa HD-1 Analyzer an ultrasensitive assay with antibodies P217 and Tau 12 was developed which could detect very low levels of P217 in brain, CSF and in blood. Variation of P217 levels was also found in different Taupathies. The Taupathies with the highest levels of P217 was Progressive supranuclear palsy, Corticobasal Degeneration and Globular glial Taupathies.