Vitamin K is a fat soluble compound which is synthesized by the gut microbiota and produced in many tissues within the body. Considering its role in the liver as a cofactor for gamma carboxylase enzymes, treatment of dark circles and pigments under the eye among others. It is clear that is some circumstances vitamin K has to cross biological barriers, particularly, when the vitamin is produced by microbiota in the intestine or applied topically on skin. Thus it is important to develop methods that allow studies of vitamin K permeability through the skin including its participation in redox reactions and transdermal permeability. Taking into account that transdermal permeability is strongly limited for high molecular weight compounds, i.e., compounds with higher than 500Da, the study was conducted with vitamin K of  lower molecular weight. Specifically vitamin K4 model, i.e., 1,4-dihydroxy-2 naphthoic acid, with molecular weight of 204g/mol. Vitamin K4 is suitable for this kind of study , because it can work as reducing (antioxidant) compound as well as has relatively beneficial physicochemical characteristics for transdermal permeability. Permeability studies were conducted with skin covered oxygen electrode and franz diffusion cell. Data from measurements were analyzed to estimate diffusion coefficients, apparent Michaelis-Menten constants and flux of a vitamin K4 model whilst contribution of different permeability pathways was determined theoretically.

Legionella omfattar bakterier som kan orsaka allvarliga lunginflammationer, såsom legionärsjuka hos människor. Den mest kända sjukdomen som kan orsakas av Legionella-bakterier är legionärsjuka, en allvarlig typ av pneumoni, vilket utgörs oftast av Legionella pneumophila (L. pneumophila). L. pneumophila kan delas in i olika serogrupperbaserat på variationer i dess yttre strukturer. Majoriteten av L. pneumophila framkallas av serogrupp 1. I Region Skåne utgörs diagnostiken med PCR analys för att detektera Legionella bakterier, som utförs på nedre luftvägsprov (Bronkoalveolärt lavage, förkortad BAL, och sputum). PCR-positiva prover odlas för att artbestämmas genom riktad odling på fasta substrat för att därefter försöka matcha Legionella stammen som isolerats från miljöer med patienten genom genetisk typning. Då odling av Legionella stammen för artbestämning vanligtvis ej svaras ut förens efter 10 dygn kan utvärdering av en PCR förenkla särskiljning av serogrupp 1 och serogrupp 2-14 för att kunna behandla patienten i tid. Wzm-PCR:en anses kunna särskilja serogrupp 1 från serogrupp 2-14. Genom att odla fram L. pneumophila serogrupp 1 isolat och därefter utföra en spädningsserie kunde PCR-effektiviteten med hjälp utav qPCR beräknas vara 96,5 %. L. pneumophila serogrupp 2-14 odlades även ut för att kunna avskilja serogrupp 1 från serogrupp 2-14 vilket gavs till resultat, men vidare testning av metoden behövs.

Tuberculosis is an airborne infection caused by Mycobacterium tuberculosis, primarily affecting the lung. Multidrug – resistant tuberculosis (MDR – TB) develops due to inadequate treatment initiation and is challenging to treat because the bacteria has developed resistance to the most effective anti – TB drugs. Conventional oral administration of Clofazimine (CLZ) often causes adverse effects like skin discoloration, increasing the risk of premature treatment interruption. Pulmonary drug delivery with mesoporous silica particles (MSP) offers a promising solution for treating MDR – TB by reducing side effects and improving patient compliance. This study aimed to investigate how different properties of MSP I, II, and III affected CLZ’s in vitro dissolution in simulated lung fluid (SLF). Additionally, the study aimed to determine if released drug concentration exceeded the minimum inhibitory concentration (MIC) used in MDR – TB treatment. CLZ – loaded MSPs at a concentration of 25 mg/l underwent dissolution testing using the sample and separate (SS) and dialysis membrane (DM) methods. Results showed that CLZ released from the MSPs exhibited similar average maximum concentration and precipitation patterns with the SS method. ANOVA showed a statistically significant higher concentration of released CLZ from MSP I compared to MSP III. This implied that the properties of the MSPs affected CLZ’s dissolution. The DM method resulted in non – quantifiable levels of CLZ. Scanning electron microscopy confirmed the degradation of the MSPs in SLF from micron to nano size and revealed differences in degradation due to the particles’ different t50%. Furthermore, using a concentration of 25 mg/l was inadequate for achieving CLZ concentrations exceeding the MIC. This implied that CLZ would be unable to inhibit the growth of the drug – resistant strains of M. tuberculosis.

α1-microglobulin (A1M) is a heme and free radical binding antioxidant found in most tissues. A1M is encoded by the alpha-1-microglobulin/bikunin precursor (AMBP) gene together with the protease inhibitor bikunin. A knock-out (KO)-model of A1M was recently established and published, however, until now it has not been utilized to explore A1M’s role in perinatal brain development. Thus, this study aims to explore the role of A1M in perinatal brain development, and its impact on the transition into a higher oxygenated environment at birth, by comparing gene expression of oxidative stress, inflammation, development, and antioxidant targets using the A1M KO-mouse model. The study design included comparisons of wild type (WT) and homozygote (HO) mice at four timepoints distributed between gestational age 19.5 and postnatal day 10. To study the gene expression, RNA was extracted from brain tissue and cDNA generated, and subsequently the cDNA was analyzed using qPCR and SYBR Green detection. 

In the study, we found that A1M is expressed at a low level in the brain. Another interest of the study was to investigate the oxidative stress related to transitioning from a low oxygenate environment in utero, to a more oxygenated environment following birth. We observed no significant fold-change differences at birth compared to other timepoints in any of the targets studied. Importantly, a potential limitation in the current study is small sample size groups (n=5 in 4 timepoint groups for each genotype). In conclusion, A1M does not seem to play a significant role in neonatal brain development or transition into an oxygenated environment under healthy conditions. However, further studies are encouraged. 

Malignt mesoteliom är en ovanlig och aggressiv cancerform. Den är vanligast i pleura och kallas då pleuralt mesoteliom. Vanligaste orsaken till utveckling av pleuralt mesoteliom är exponering för asbest. Insjuknande patienter har dålig prognos och begränsade behandlingsmöjligheter. Röntgen kan i ett första stadie ge diagnos av sjukdomen. Oftast behövs också en biopsi tas för fastställande av diagnos. Även provtagning av pleuravätskan kan ge värdefull diagnostisk information. Immunhistokemi är en viktig tilläggsanalys för diagnos med biopsier och cellblock. Immunhistokemi innebär färgning med antikroppar. För pleuralt mesoteliom kan exempelvis antikropparna PD-L1 och calretinin användas för diagnostisering. I denna studie färgades 10 parade pleurabiopsier och cellblock från pleuravätska från patienter med malignt mesoteliom. Antikropparna PD-L1 och calretinin användes. Syftet var att utvärdera uttrycket av PD-L1 och calretinin. Alla infärgade glas undersöktes i ljusmikroskop. För PD-L1 ansågs enbart membranfärgning som positivt infärgad och för calretinin ansågs cytoplasmatisk och/eller kärnfärgning som positiv. Procentuellt antal positiva tumörceller undersöktes vid två cutoff värden, ≥1 % och ≥10 %. Procentuellt antal <1 % vid cutoff ≥1 % och procentuellt antal <10 % vid cutoff ≥10 % ansågs negativt. Detta gällde för båda antikropparna. Antal positiva och negativa biopsier samt cellblock och konkordansen redovisades. Overall percentage agreement (OPA), Cohen’s kappa (κ) och konfidensintervall (CI) beräknades också. Resultatet visade på lägre antal positiva färgningar för cellblocken jämfört med biopsierna för båda antikropparna. För konkordansen visades att den vara lika för PD-L1 och calretinin vid cutoff ≥1 % men högre för PD-L1 än för calretinin vid cutoff ≥10 %.

Nano-meter sized particles of de-aluminated zeolite Y has a high adsorption capacity of both low molecular weight bio-molecules and macromolecules. In this study we used de-aluminated zeolite Y as a novel approach to study the mechanisms of endocytosis in immature human peripheral blood dendritic cells (DCs). Probes detecting pH neutral and acidic endosomes were adsorbed to the zeolite and used as a tracer of the endosomal pathway of a cell in the form of acidification and lysosomal function. Both the myeloid (M-DCs) and the plasmacytoid (P-DCs) dendritic cell subsets showed an endocytosing capacity comparable to peripheral blood monocytes but only the M-DCs were able to form acidic endosomes after internalization of zeolite particles. Furthermore, during lipopolysaccharide (LPS) stimulation of the DCs population, an enhanced induction of acidic endosomes was only seen in the M-DC population. Proteolytic degradation of endocytosed proteins was detected using self-quenched DQ-ovalbumin adsorbed to zeolite particles and our results showed a clear difference between the two DC populations. The M-DC population, that showed formation of acidic endosomes, also showed proteolytic degradation of ovalbumin. The P-DC population on the other hand, showed no formation of acidic and no proteolytic degradation of ovalbumin. Various bio-molecules can be adsorbed by de-aluminated zeolites and in conclusion we propose the use of zeolite particles as a useful tool in the study of the endocytosing mechanisms of a cell.

The adsorption from 0.2% (w/w) chlorhexidine and black tea solutions onto an in vitro pellicle from whole unstimulated saliva on hydroxyapatite discs was studied by ellipsometry. It was found that chlorhexidine adsorbed to the pellicle causing a modification of the pellicle properties, leading to a subsequent increase in adsorption of salivary and black tea components. There was a distinct order of addition effect, whereby chlorhexidine followed by black tea gave an overall greater adsorption of components compared to black tea followed by chlorhexidine. This increase in adsorption gave a concomitant increase in colour or stain as measured by a reflectance chromameter. The increase in adsorbed amounts and stain was modified in part by the adsorption of salivary fractions between the chlorhexidine and black tea treatments. In all cases, the chlorhexidine and black tea modified pellicles were not readily removed by either phosphate or sodium dodecyl sulphate rinses. Thus, following chlorhexidine exposure, the accelerated adsorption of salivary and black tea components can ultimately lead to increased staining of the pellicle.

The adsorption of black tea and red wine components onto a pellicle-like protein layer formed in vitro by adsorption from whole unstimulated saliva on hydroxyapatite discs were studied by in situ ellipsometry. It was found that components from black tea readily adsorbed to the pellicle. Subsequent exposure to saliva led to further adsorption of salivary components to give an overall increase in the amounts adsorbed. The amounts adsorbed increased still further following a third tea and saliva exposure. Components of red wine gave significantly greater amounts of adsorption to the pellicle than black tea. The adsorption of components of black tea gave a concomitant increase in colour or stain as measured by a reflectance chromameter. In all cases, the black tea- and red wine-modified pellicles were not eluted by either phosphate buffer or sodium dodecyl sulphate (SDS) rinses. Thus, black tea and red wine components have been shown to have a profound effect on in vitro pellicle maturation, causing thickened layers of stained material to build up, which are not readily removed.

Competitive adsorption between B-casein and B-lactoglobulin (B-Lg) during spray-drying was studied by the new surface sensitive technique using fluorescence quenching of pyrene labelled protein at the powder surface. The difference in competitiveness of B-casein when present as monomers and as associated into micellar like structures were studied. Results were compared with the adsorption of single proteins at the powder surface. The adsorption of monomeric B-casein alone gave an apparent surface load of 1 mg/m2 at a protein concentration of 0.3% dry weight and then remained constant with an increasing protein concentration. In the presence of Ca2+, associated B-casein gave a lower affinity adsorption than monomeric B-casein and did not reach a plateau value, instead it continued to increase with an increasing protein concentration. B-Lg showed a low-affinity adsorption during spray-drying compared to monomeric B-casein, although not as low as associated B-casein. Competitive adsorption between monomeric B-casein and B-Lg resulted in a higher apparent surface load of B-casein than B-Lg at both protein concentrations studied (total 0.3 and 3.3% dry weight). However, in an associated form B-casein was less competitive than B-Lg. At a low bulk protein concentration (0.3% dry weight) B-Lg dominated the powder surface, whereas at a higher concentration (3.3% dry weight) there was little difference between the proteins. The results indicate that the competitiveness of a protein during spray-drying is highly influenced by the ability of the protein to attach and rearrange at the droplet's air?water interface during the spray-drying process.

Due to a growing demand and need for faster results, effectivization as a concept has been pushed to the forefront of medical research. Here, a method transfer between 2 fully automized ELISA instruments is highlighted. The instruments were the Tecan Freedom EVOlyzer and the Dynex DSX.  The Tecan instrument is a more modern instrument with a more extensive LIMS integration, better automatization and increased capacity for samples and assays. The method being transferred was an Anti-GAD ELISA for the diagnosis of Type 1 Diabetes Mellitus. The study design was a result comparison of 20 samples with varying Anti-GAD concentration and 15 replicates of a single sample for a precision study. To successfully transfer the method, acceptance criteria concerning percentual difference in result, intermediate precision, and repeatability had to fall below 20 %. The assay was set up as a sandwich ELISA in accordance with the kit used at the laboratory (Euroimmun), where each sample was set as duplicates. Preliminary programming and testing was performed to ensure proper function. An average bias of +33 % was reported, as was an intra-run variation and an inter-run variation of ca 11 %. Additionally, a recurring issue with samples and calibrators in specific locations on the plate was reported. The issue was deemed systemic and possible solutions include changing programming parameters, or instrument part replacement. Ultimately the method transfer was left incomplete, and several technical details must be overcome before taking Anti-GAD analysis into routine use on the instrument.

The aim is to illuminate aspects of dental hygienists' work in multiethnic/multicultural societies, with focus on the intersection between the institutional activity and migration, ethnic diversity and culture issues. Hence, a tension between equality and diversity are discussed. The thesis has two analytical approaches, practice and discourse. The focus is on talk-in-interaction with patients (practice) and how the dental hygienist conceptualise their work when treating people with migrant background (discourse). The theoretical and methodological considerations are drawn from the sociological tradition of "Verstehen", Foucault and discourse analysis. The empirical material is based on documented observations (VCR) and interviews with dental hygienists and patients. The analysis of practice suggests that the interaction is organised by the institutional activity type. A patient’s readiness to position oneself as a "knowing subject", opposed to ethnicity, is crucial to how the interaction is structured. Migration related categories and phenomenon are related to the institutional task and understood as situated including practices. In discourse, ethnicity intersects with gender and class. The patient's power resources are of vital importance in a discourse on "immigrant patients". The hygienists relates to culture, in terms of relationship with dental care. References in exclusionary discourses were also found. Based on multilayered findings and theories of multiculturalisms, the concept of culture is discussed in connection to agency, ethics of care and equal treatment.

ABO- och Rh-systemet är de mest kliniskt betydelsefulla blodgruppssystem för nutidens serologiska diagnostik. En viktig analysmetod inom detta område är kvantifiering av blodgruppsantikroppar då mängden antikroppar i plasma bestäms. Denna metod har flera olika användningsområden, bland annat för att bestämma mängden av irreguljära antikroppar för gravida vid immunisering då mamman har bildat antikroppar mot ett antigen som fostret har. Metoden utförs även i samband med transplantationer och för trombocytgivare. Kvantifiering av blodgruppsantikroppar kan utföras både manuellt och automatiskt. Den manuella metoden utförs med diluent pH7, en form av fosfatbuffrad saltlösning (PBS), som spädningsvätska, medan den automatiska utförs med en dyrare titreringslösning. Ett mer kostnadseffektivt val skulle vara att använda diluent pH7 som spädningsvätska för den automatiska metoden. Denna studie är en metodoptimering för automatisk titrering av blodgruppsantikroppar med IH-500 instrument. Plasmaprover från tre olika kategorier av individer (transplantationspatienter, trombocytgivare och gravida kvinnor) analyserades på IH-500 instrument för både ABO- och andra blodgruppsantikroppar. I maskinen utförs en spädningsserie av plasma från patienter och efter tillsättning av testerytrocyter observeras var det skett en agglutination och hur starkt den var. Det första spädningssteget som graderas som 1+ reaktion räknas som antikroppstitern. Resultat redovisades i form av stapeldiagram som visar skillnaden i antikroppstiter med både titreringslösning och PBS som spädningsvätska. Samtliga resultat ligger inom ramen för vad som definierats som ett godkänt resultat. Konklusionen av studien är att PBS är ett effektivt och billigare alternativ som kan användas som spädningsvätska vid automatisk titrering av ABO- och andra blodgruppsantikroppar på IH-500 instrument.

Salivary pellicles display exceptional hydration and lubrication performance. At present, there are still gaps in the understanding of how this is achieved. The aim of this thesis was therefore to increase our knowledge on the mechanisms underlying these properties and deepen the understanding of how they are related to the composition and structure of pellicles, with a focus on those formed under in vitro conditions. This has applications ranging from the development of artificial saliva and lubricating coatings for biomedical applications to methodological approaches for initial testing of oral healthcare products. For this, we also focused on developing suitable methodological approaches for these studies, centering on atomic force microscopy, quartz crystal microbalance with dissipation monitoring, ellipsometry and neutron reflectometry techniques, to investigate in vitro and model salivary pellicles.

First, we confirmed a two-layer structure for in vitro salivary pellicles and showed that the outer layer is mainly composed by the oral mucin MUC5B, but that it also contains other salivary components that enhance swelling and hydration. In the presence of bulk saliva, the outer layer also contains a reversibly and loosely bound fraction. This fraction increases the adhesiveness of the pellicle but unexpectedly has no significant effect on its lubrication performance. We also investigated the effect of mechanical confinement on model salivary pellicles by means of Neutron Reflectometry, revealing that at a pressure of 1 bar they are already completely compressed and dehydrated. Finally, with the aim to advance towards better oral healthcare products, we investigated the effect of nonionic and amphoteric surfactants on salivary pellicles, showing that they have a gentler effect on pellicle structure than the commonly employed anionic surfactants.

Vasopressin är ett viktigt hormon som har många fysiologiska funktioner, däribland upprätthållandet av vätskebalansen i kroppen. Mätning av detta hormon är dock komplicerat och därför används ”skuggfragmentet” copeptin, som härstammar från samma prekursor. Genom användandet av metoden B·R·A·H·M·S KRYPTOR compact PLUS mäts copeptin. Studiens syfte är att beräkna variations-koefficienten och därmed undersöka de uppmätta värdenas reproducerbarhet. Vidare blir syftet att använda EpiHealth-kohortstudien för att validera den redan kända kopplingen mellan copeptin och förhöjt blodsocker genom en multivariant linjär regression. Vi kan i arbetet konstatera att copeptin metoden har en god reproducerbarhet, där majoriteten av de multipelt uppmätta copeptin-värdena har en inter-assay CV <8%. Vid undersökning av EpiHealth-kohorten fann vi att en ökning av copeptin var kross-sektionellt associerad med ett flertal metabola riskmarkörer, däribland fastande plasma-glukos, efter multivariant justering. Att copeptin var signifikant relaterat till denna potenta metabola riskmarkör kan tyda på att det finns ett orsakssamband mellan förhöjt vasopressin och förhöjt blodsocker, något som även tidigare studier har pekat på. Detta i sin tur visar att vasopressin kan spela en roll i utvecklandet av typ 2-diabetes. Om ett orsaks-samband föreligger undersöks nu i en stor randomiserad klinisk studie där vasopressin-nivåerna hos hälften av deltagarna sänks med hjälp av ökat vatten-intag (H2O-metab-studien). Det finns förhoppningar om att användandet av copeptin skall kunna användas i klinisk verksamhet för att riskbedöma individer avseende kardiometabola sjukdomar (däribland typ 2-diabetes).

Diabetes has, in the past three decades, surged immensely. Because of this, new insulin analogues are constantly in the making. 

In clinical studies, the presence of antidrug antibodies can prove a challenge when measuring insulin. In order to overcome the interference from antidrug antibody complexes on the total insulin measurement in human serum, several pre-treatment methods on insulin and polyclonal antibodies spiked samples were tried using ELISA analysis.

Several different methods were tried, acid dissociation using a glycine buffer with and without ethanol in different concentrations, high ionic strength dissociation using MgCl₂, Polyethylene glycol (PEG) and filtration.

The best results were found when using the acid dissociation technique. Using glycine promising results were achieved, especially when 20 % ethanol was added to the acid mixture. Pre-treatment using PEG, MgCl₂ and filtration was unsuccessful with the methods used.

The main goal was reached through the use of glycine with the addition of 20% ethanol for acid dissociation. The proposed method still leaves significant room for optimisation and needs further verification on real patient samples. However, it is a good step in the direction of a global methodology using ELISA to overcome antidrug antibody interference for total insulin measurement in human serum.

ISOLERING OCH KVANTIFIERING AV GENOMISKT DNA FRÅN MAKROFAGER STIMULERADE MED ALUMINIUMADJUVANT

GECHSIEM CHUY

Chuy, G. Isolering och kvantifiering av genomiskt DNA från makrofager stimulerade med aluminiumadjuvant. Examensarbete i biomedicinsk laboratorievetenskap, 15 högskolepoäng. Malmö universitet: Fakulteten för hälsa och samhälle, Institutionen för biomedicinsk laboratorievetenskap, 2022.

Makrofager har flera funktioner i immunförsvaret och förekommer i olika fenotyper beroende på stimulering. M1 makrofager är pro-inflammatoriska och stimuleras av lipopolysackarider och interferon-gamma. M2 makrofager är anti-inflammatoriska som stimuleras av interleukin-4 (IL-4) och IL-13 medan M0 makrofager är icke polariserade. Dessa olika former av makrofager utför olika funktioner och behöver därför uttrycka olika delar av genomet. Reglering av genuttrycket sker bland annat genom metylering av cellernas DNA. Aluminiumadjuvant har under lång tid använts som tillsatser i vaccin och fungerat som förstärkare för immunförsvaret mot antigenet som ingår i vaccinet. För att kunna undersöka om aluminiumadjuvant också påverkar makrofagers metyleringsgrad måste genomiskt DNA kunna isoleras från cellerna både med och utan stimulering av aluminiumadjuvant. Syftet med studien var att undersöka om differentiering och polarisering av makrofager i cellodlingsplattor med bottenytor av 3,8 respektive 9,5 cm2 i brunnarna ger nog med celler för att reproducerbart kunna isolera och kvantifiera genomiskt DNA med en koncentration av minst 1 ng/μl från makrofager stimulerade med aluminiumadjuvant. Resultatet från denna studie visar att differentiering och polarisering av makrofager i cellodlingsplatta med bottenyta 9,5 cm2/brunn ger tillräckligt med celler för att kunna extrahera genomiskt DNA med koncentrationer högre än 1 ng/μl. Betydligt högre mängder DNA erhölls när cellerna eluerades från cellodlingsplattan innan cellerna lyserades inför isolering DNA. Det är därför av största vikt att adherenta celler elueras och att lysering av celler inför isolering av genomiskt DNA görs på celler i suspension. Vidare visade resultaten att för makrofagerna som stimulerade med aluminiumadjuvant gav lägre DNA utbyte jämför med cellerna utan stimulering. Studien har visat ett förfarande för optimering av preparationssteg inför DNA extraktion som gör det möjligt att isolera DNA på ett reproducerbart sätt inför fortsatta studier.

Nyckelord: Aluminiumadjuvant, DNA, immunförsvaret, makrofager, polarisering.

Non-invasive healthcare technologies are increasingly pivotal in research anddevelopment due to their affordability and the convenience they offer to bothhealthcare recipients and providers. Alongside traditional non-invasive methodssuch as ultrasound imaging, a variety of innovative non-invasive devices havebeen developed. These include cardiovascular diagnostic systems, bioimpedancebasedscales, and various types of analyzers. These analyzers, which can be fluidlessor fluid-based, are capable of measuring not just physical parameters of thebody but also key biomarkers like glucose and lactate. This comprehensive andtransdisciplinary thesis encompasses three distinct yet interconnected segments:1) Advanced ultrasound imaging (Papers I and II): The first explored vortexformation time in female athletes and the second detailed investigations of thesuperficial venous systems of apparently healthy volunteers.2) Validation and application of commercially available fluid-less bloodanalyzers (Papers IV-VI). These papers focus on non-invasive blood glucosemonitoring (Paper IV) and the general use of non-invasive healthcaretechnologies among female participants from socioeconomicallydisadvantaged areas (Papers V and VI).3) Design and testing of novel, fluid-based sensors, and biosensors (Papers II andIII): Paper II delves into biosensing of viruses, and paper III deals withcontinuous ex vivo glucose sensing in human blood using an enzymatic sensorin a vein replica.Each of these segments contribute to the broader understanding and advancementof non-invasive healthcare technologies, highlighting the significant role suchtechnologies play in modern healthcare research. The thesis's transdisciplinaryapproach, spanning from advanced imaging techniques to the development ofnovel biosensors, exemplifies the dynamic and evolving nature of medicaltechnology research.

The contribution of nonspecific interactions to the overall interactions of thiol-ssDNA and dsDNA macromolecules with gold nanoparticles was investigated. A systematic investigation utilizing dynamic light scattering and cryogenic transmission electron microscopy has been performed to directly measure and visualize the changes in particle size and appearance during functionalization of gold nanoparticles with thiol-ssDNA and nonthiolated dsDNA. The results show that both thiol-ssDNA and dsDNA do stabilize gold nanoparticle dispersions, but possible nonspecific interactions between the hydrophobic DNA bases and the gold surface promote interparticle interactions and cause aggregation within rather a short period of time. We also discuss the adsorption mechanisms of dsDNA and thiol-ssDNA to gold particles.

Mitochondria are important cytosolic organelles present in nearly all eukaryotic cells. The main function of mitochondria are to generate the vast majority of ATP through the process of oxidative phosphorylation. Mitochondria have key roles regarding other systems in the body as well, such as regulation of apoptosis, calcium homeostasis, reactive oxygen production etc. Mitochondrial diseases are caused by impaired mitochondrial function, originating from mutations in either the mitochondrial DNA or the nuclear DNA. Epilepsy is a common symptom of mitochondrial disease, especially in children. The pathophysiology behind mitochondrial epilepsy is primarily based on ATP deficit, leading to a negative effect on a range of different nervous system related functions that in the end leads to seizures. The study aimed to investigate the effect on mitochondrial respiration of two commonly used antiepileptic drugs, namely valproic acid and Topiramate, and the anesthesic drug propofol, commonly used in case of refractory status epilepticus. The three drugs were titrated in different concentrations in a high-resolution respirometer from Oroboros Instruments (n=6). Propofol seemed especially inhibiting of mitochondrial function, and both propofol and topiramate had a significant decrease in mitochondrial respiration within the clinical concentrations. The result of the study supported research stating that propofol should be used with caution in patients with a mitochondrial disease, but further research should be done regarding all three drugs in order to draw definite conclusions.

Vid organtransplantation finns det en möjlighet att transplantera organ över blodgruppsgränserna, det vill säga att ett organ transplanteras mellan individer med olika blodgrupper. Det är i dag en etablerad teknik som ökar möjligheten till fler transplantationer. Innan en sådan transplantation kan ske måste mottagaren genomgå en behandling som går ut på att manipulera immunsystemet samtidigt som de inkompatibla blodgruppsantikropparna extrakorporealt minskas till en given nivå hos recipienten före transplantation. Behandlingarna görs vanligtvis konsekutivt av recipienten tills det att en för transplantation accepterad antikroppsnivå underskrids (titer<1:8) och görs för att minimera risken för bortstötning av det transplanterade organet. För att eliminera de inkompatibla blodgruppsantikropparna är affinitetskromatografi genom interaktioner mellan antikroppar och antigener en lovande separationsmetod. Syftet med arbetet var att jämföra upptag av specifika antikroppar från en given plasmavolym genom att upptaget via en kontinuerlig adsorptionsprocess mot fraktionerad adsorption studerades. Syftet undersöktes genom att testa två olika immobiliserade A-trisackarider. Albuminförlusterna undersöktes även för att se om någon metod tog bort mer proteiner från plasman då det kan ha stor betydelse för patientens blödningsbenägenhet. Resultatet visade inte på någon skillnad mellan kontinuerlig metod jämfört med fraktionerad metod och ytterligare undersökning måste göras för att konstatera vilken metod som skulle vara att föredra.

Vid Klinisk kemi och farmakologi, Specialkemi, vid Skånes universitetssjukhus, Lund, utförs kvantifiering med LC-MS/MS av antipsykotiska och antidepressiva läkemedelskoncentrationer i serum med acetonitril (ACN) som mobilfas. ACN är på grund av sin höga elueringsstyrka ett av de vanligaste organiska lösningsmedlen vid reversed phase (RP) kromatografi, men uppvisar samtidigt en hög toxicitet med risk för stora leveransproblem. I syfte att reducera mängden ACN undersöktes därför möjligheten till ett mobilfasbyte till metanol (MeOH). Ordinarie metod jämfördes med tre nyutvecklade metoder med MeOH-baserad mobilfas. I en av metoderna ändrades endast mobilfas och elueringsgradient, medan två av metoderna även använde andra sorters RP-kolonner med anpassade elueringsgradienter. Samtliga analyter uppvisade godkänd separation och retention vid eluering med MeOH, men stor fluktuation från referensmetod sågs vid kvantifieringen av flera analyter, däribland olanzapin, desmetylolaznapin och mirtazapin. Liknande avvikelser med avseende på regression och kvantifieringsdifferens observerades vid eluering med andra sorters RP-kolonner. Detta indikerar att vidare optimering av andra vätskekromatografiska och masspektrometriska parametrar bör utföras innan metoderna kan valideras.  

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is the virus that causes coronavirus disease-2019 (COVID-19). The virus was first identified in December 2019, spread worldwide, and caused many deaths. The World Health Organization (WHO) declared a global public health crisis on 30 January 2020 and a pandemic on 11 March 2020. SARS-CoV-2 is a single-stranded RNA virus belonging to the genus Coronavirus and the family Coronaviridae. SARS-CoV-2 has four structural proteins, Envelope (E), Membrane (M) and Nucleocapsid (N) and Spike (S) proteins. The S protein consists of S1 and S2 subunits. The receptor-binding domain (RBD) of the virus is located in the S1 subunit and binds the virus to the surface receptor angiotensin convertase-2 (ACE2) of the host cell. This study aimed to better understand the production of proinflammatory cytokines in macrophages after in vitro interaction with the RBD of SARS-CoV-2. Gene expression of tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6) and interleukin-1 Beta (IL-1β) in RBD stimulated mouse macrophage cell line RAW 264.7 was detected with quantitative Reverse Transcription Polymerase Chain Reaction (RT-qPCR). In addition, the concentration of secreted TNF-α, IL-6 and IL-1β was quantified with sandwich enzyme-linked immunosorbent assay (ELISA). The study indicated that mouse macrophages produce TNF-α upon exposure to SARS-CoV-2 RBD after 6 and 24 hours of cell stimulation, but neither IL-6 nor IL-1β were increased after stimulation. The future goals of the study are to understand how the immune system responds to the SARS-CoV-2 infection, and how this can help in treating COVID-19.

 Background: Niacinamide (NIA) is one of the most commonly used cosmetic ingredients. It belongs to the vitamin-B3 family and has extensive dermatological therapeutic benefits. NIA has been proven to be a useful skincare product in serving as anti-acne agent, preventing skin hyperpigmentation, removal of wrinkles from the face etc. 

Aim: To investigate permeability patterns of NIA, its effect on electrical impedance of the skin membrane and the role it plays in maintaining the hydration of stratum corneum (SC). For this, permeation, chromatography, sorption isotherm and X-ray studies were performed.

Results: NIA permeation was observed to correlate with pH and it permeated more when delivered in PBS at pH 7.4 as compared to its permeation in citrate buffer at pH 5. Moreover, skin resistance also increased by Ca. 47% in relation to NIA permeation at pH-5 while it decreased by an average of 45% at pH 7.4. In addition, vapor sorption analysis showed that NIA increased the hydration of SC at 95%RH as compared to buffer controls. This was also supported by X-ray data where NIA treated SC samples were shown to have larger interchain spacing in their keratin filaments in comparison to SC in buffer controls. This increase is usually associated with an increase in the water content of SC and thus NIA might have similar beneficial effects as water and can even be more advantageous as it doesn’t evaporate in dehydrated states unlike water. Moreover, artificial skin model has also been tested in parallel, and it was significantly more permeable to NIA than the human skin. Hence some modifications are necessary before it can be used to replace human/porcine skin.

Conclusion: The study showed that pH influences NIA permeation and resistance of skin membrane. Additionally, NIA play beneficial roles by increasing water content of SC at high relative humidity (RH%).

Medfödd immunitet är en viktig del av immunsystemet och omfattar, bland annat, mononukleära fagocyter. Det mononukleära fagocytsystemet klassificeras i två stora funktionella grupper, M1 makrofager och M2 makrofager samt den opolariserade formen, M0 makrofager. Olika makrofager har olika vitala funktioner. Alveolära makrofager, koloniserar lungvävnaden redan från födseln och är oberoende av andra vävnadsmakrofager och monocyterna som förekommer i blodomloppet. Immuncellerna i lungan måste upprätthålla dämpad aktivitet för att utesluta överdrivna reaktioner med efterföljande skador på lungvävnaden. Syftet med detta projekt var att bekräfta och kvantifiera förekomsten av osteopontin, IL-28 A/B och CCL6/C10 i medier från alveolära makrofager och jämföra detta med cytokinproduktionen från makrofager, differentierade och polariserade från mjält-, och benmärgsceller. Kvantifiering av cytokinproduktionen utfördes med ELISA kit på ostimulerade medier från benmärg, mjälte och lunga samt stimulerade med MPLA eller aluminiumadjuvant. Analysen bekräftade förekomsten av osteopontin och CCL6 medan IL-28 inte förekom. Vidare visades likheter i cytokinproduktion mellan alveolära makrofager, M1 makrofager från benmärg och M2 makrofager från mjälte. Paralleller dras bland annat mellan alveolära makrofagers uttryck av ytmarkörer som härrör från M1 samt M2 makrofager och cytokinproduktionen.

Portabel medicinteknisk utrustning framträder alltmer som en av de mest lovande metoderna för vårdövervakning och personlig behandling. Förebyggande vård och hantering av kroniska sjukdomar är resurskrävande och en överföring av det konventionella sjukhuscentrerade sjukvårdssystemet till ett individcentrerat vårdsystem skulle vara samhällsekonomiskt gynnsam. I ett sådant scenario representerar bärbara mätenheter en teknik för övervakning av patienter på ett icke-invasivt och lättanvänt sätt. Denna teknik har möjlighet att tillhandahålla långsiktiga hälsostatusövervakningar och förmedla realtidsdata som läkare kan analysera för att ge patienterna återkoppling utan att behöva träffa patienterna lika ofta. Dessutom är många utan kroniska sjukdomar också intresserade av att övervaka kroppens hälsotillstånd för att förhindra sjukdomar och uppnå en högre livskvalitet. Dagens bärbara enheter integrerar elektronik med låg strömförbrukning och trådlös teknik, s.k. ”low power wireless technology”, för att överföra information från enheten till en mottagare. Elektronik behöver tillförlitliga strömkällor för att säkerställa funktionen, och biologiska kraftkällor är särskilt lämpliga alternativ att använda i bärbara enheter, eftersom de har hög prestanda när de används under fysiologiska förhållanden. Olika biologiska kraftkällor har tillverkats och testats i denna avhandling. Materialen som används för att tillverka dem är transparenta och flexibla. Dessa två egenskaper bidrar starkt till användarvänligheten och ökar därmed benägenheten att använda sådana kraftkällor. De biologiska kraftkällorna omvandlar kemisk energi till elektrisk energi genom att oxidera glukos och reducera syre under förhållanden som liknar dem som föreligger i mänsklig tårvätska. Detta arbete bidrar till att öka kunskapen om flexibla, transparenta och nanostrukturerade material som används för tillverkning av biologiska kraftkällor.

The role of saliva in the oral cavity is manifold; an important function is to serve as lubricant between hard (enamel) and soft (mucosal) tissues. Intraoral lubrication is of crucial importance in order to maintain functions such as deglutition, mastication and the faculty of speech. A large number of people suffer from impaired salivary functions, displaying symptoms such as 'dry mouth'. This results in a need for methods to assess the lubricating properties of both native saliva and potential artificial saliva formulations. Here, normal as well as lateral forces, acting between adsorbed salivary films, have been measured for the first time by means of colloidal probe atomic force microscopy (AFM). It was found that the presence of salivary pellicles between hard surfaces reduces the friction coefficient by a factor of 20. This reduction of friction is consistent with the long-range purely repulsive nature of the normal forces acting between the salivary films. The lubricating mechanism is presumably based on a full separation of the sliding surfaces by the salivary films. The friction between salivary films has been investigated at normal loads that cover the clinical jaw closing forces, and it can be concluded that the lubricating properties are maintained within this load interval. The present study indicates the usefulness of colloidal probe AFM, which offers a direct and quantitative measure of lubrication at a molecular level, in the study of biotribological phenomena. In particular, the results obtained here may have implications for the development of saliva substitutes.

EARLY ACTIVATION MARKERS EXPRESSED BY HUMAN PERIPHERAL DENDRITIC CELLS Peter Hellman and Håkan Eriksson University of Malmoe, Department of Biomedical Laboratory Science E-mail address: peter.hellman@hs.mah.se Two major populations of immature dendritic cells, myeloid (M-DCs) and plasmacytoid (P-DCs) can be identified in human peripheral blood. Activation of these subsets through their Toll-like receptors (TLRs) (TLR4 for M-DCs and TLR9 for P-DCs) induced production of the chemokine Il-8, already within two hours of stimulation. The production of IL-8 preceded the expression of the activation marker CD40 in both M-DCs and P-DCs. Although both populations of DCs secreted Il-8 upon activation, the levels of Il-8 produced was several times higher in the M-DCs compared to the P-DCs population. Before activation both subsets of DCs expressed the IL-8 receptor type B (CD128b), however, upon stimulation the Il-8 receptor became undetectable in both M-DCs and P-DCs. Increased expression of MHC class II molecules is regarded as an early activation marker of DCs. However, only the P-DCs showed a significantly increased expression of MHC class II after 4 hours of stimulation through TLR9. Noteworthy, the M-DCs showed an unexpected increase of MHC class II molecules after conditioning in medium for 4 hours, and no further increase in MHC class II expression after stimulation through TLR4 was observed. In conclusion, we propose that during activation of human DCs the production of Il-8 and loss of CD128b are the earliest signs of activation preceding both MHC class II, CD40, CD80 and CD86 expression.

Tuberculosis is a fatal airborne disease caused by bacteria from the Mycobacterium tuberculosis complex (MTBC). The incidence of contracting tuberculosis is estimated to be around 10.6 million cases each year. Increased drug resistance among mycobacteria has led to the need to develop new treatments. The study's purpose was to determine the antimycobacterial ability of drug complexes and how toxic these complexes are against human cells. Drug complexes from "phage derived endolysins" and "A pyrazine amide-4 aminoquinoline hybrids" were studied to possibly be included as a treatment against tuberculosis in the future. The minimum inhibitory concentrations (MIC) of the drug complexes were analyzed by the method Resazurin microtiter assay (REMA), where the results were assessed visually. The toxicity of the drug complexes was studied by growing THP1-Blue™ NF-κB cells, which then were exposed to the drug complexes. The results could then be obtained by absorbance measurement with spectrophotometry. One-way ANOVA showed a non-significant value, as the P-value was 0.44 (P>0.05). However, more supplementary studies need to be carried out to obtain a significant result. All performed concentrations of the drug complexes were assessed as non-toxic against human THP1-Blue™ NF-κB cells.

Periodontal disease ranks among the most expensive health conditions to treat, asreported by the World Health Organization (WHO). This is due to the fact thatdiagnosis is based on several specific clinical criteria that employ methods suchas inspection, palpation, probing, and interpretation of radiographic images.However, since these diagnostic tools do not provide information about patientsat risk of developing severe stage periodontal disease, patients are oftenovertreated. Porphyromonas gingivalis is a prevalent bacterium in thesubgingival crevice of patients with periodontal disease and has been termed akeystone pathogen in these conditions. P. gingivalis together with its enzymes,Rgp and Kgp, is therefore of interest as potential biomarkers on which to builddiagnostic tools based on artificial receptors. Firstly, molecularly imprintedpolymers using either the native enzymes or short sequence epitopes from themcan be used to determine the expression level of the enzymes in samples.Secondly, the enzymatic activity can be determined by recording changes inelectrochemical signals before and after hydrolysis of a specially designedpeptide sequence selective for one of the enzymes. Finally, reversible selfassembledmonolayers bearing ligands specific for bacterial adhesion throughmultivalent interactions can potentially be employed to selectively separate anddetect P. gingivalis. Together, they form the foundation for designing acommercially exploitable biosensor that combines detection methods to improvethe accuracy of diagnosis.

Vitamin B3 (niacinamide) and Vitamin B5 (dexpanthenol) are commonly used in skincare to enhance skin barrier formation, but there are currently not many studies about their effect on gene expression. This study investigated the viability of keratinocytes at different cell concentration limits (i.e., cell death). Based on the results, the cells were treated with the vitamins with the selected concentrations 1 mM and 10 mM. After the cells were treated with vitamins for 6 h, and 24 h, qPCR was employed to investigate the effects of these substances on the expression of differentiation markers, which are related to the formation of protein and lipid components required during skin barrier formation. The selected genes are related to the synthesis of filaggrin, NIPAL-4, ELOVL-4, STS, and ALOXE-3. The results showed an increase in viability at lower concentrations in cells treated with vitamins. A downregulation of filaggrin and NIPAL4 after treatment with niacinamide for 6 h and an increase of ELOVL-4 at 24h can be observed. After 6 h of incubation with dexpanthenol added, there was also a slight increase of ALOXE-3, which was more prominent upregulated at 24h. In conclusion, the vitamins seemed to increase cell viability in association with increased proliferation. This study also presented what genes were up and downregulated after treating cells with vitamins and attempted to explain how it could be interpreted.   

Antibodies have become a critical component of many diagnostic assays and are used for therapeutic purposes. Nevertheless they often fail to meet the strict performance demands raised in industry and in the clinic (e.g. stability, reproducibility, selectivity, affinity). These issues are especially notable for assays targeting post translational modifications (PTM) of proteins (phosphorylation, glycosylation, sulfation etc.). Antibody-based technologies suffer from problems of a more general nature associated with the analytical use of biological receptors i.e.: i) limited stability requiring cold chain logistics, ii) high production costs, iii) batch to batch variability. The above emphasizes the need for alternative robust, reproducible and low cost “binders” and assays. The aim in this thesis is to design, develop and test molecularly imprinted polymers (MIPs) which were synthesized epitope and stoichiometric imprinting approaches targeting phosphorylation as a PTM. Protein phosphorylationis one of the most common PTM, which is based on covalent attachment of phosphate group to particular amino acids. Misregulation of phosphorylation process is found related with diseases such as cancer, diabetes, and neurodegeneration. MIPs are synthesized through copolymerization of functional monomers and crosslinkers in the presence of N- and C- terminal protected templates. The key recognition element employed in developed synthetic receptors was 1,3-diaryl urea functionalmonomer 1. This monomer is a potent hydrogen bond donor forming strong cyclichydrogen bonds with oxyanions. Amino acid sequence specific and side chain imprinted binders were prepared targeting phosphorylation on tyrosine (pTyr) and on histidine (pHis). pHis MIP-based approach is proposed as a solution to enrich pHis peptides in the presence of other phosphoesters such as phosphoserine (pSer) in complex mixture without pre-treatment like β-elimination. In pTyr, ZAP-70 (zeta associated 70 kDa protein), which is prognosticator for chronic lymphocytic leukemia (CLL), and pTyr-sequence specific motif Src-SH2 domain were chosen as targets to evaluate regio- or stoichiometric selectivity performance of imprinted polymers. The synthesized polymers are used as effective enrichment tools for target phosphorylated peptides from complex mixture prior to mass spectrometry. Overall, the results demonstrate unique proteomics enrichment tools that link with personalized medicine relying on diagnostic coupled cancer treatment strategies based on kinase inhibitors.

BACKGROUND: Obtaining functional sperm cells is the first step to treat infertility. With the ever-increasing trend in male infertility, clinicians require access to effective solutions that are able to single out the most viable spermatozoa, which would max out the chance for a successful pregnancy. The new generation techniques for sperm selection involve microfluidics, which offers laminar flow and low Reynolds number within the platforms can provide unprecedented opportunities for sperm selection. Previous studies showed that microfluidic platforms can provide a novel approach to this challenge and since then researchers across the globe have attacked this problem from multiple angles.

OBJECTIVE: In this review, we seek to provide a much-needed bridge between the technical and medical aspects of microfluidic sperm selection. Here, we provide an up-to-date list on microfluidic sperm selection procedures and its application in assisted reproductive technology laboratories.

SEARCH METHOD: A literature search was performed in Web of Science, PubMed, and Scopus to select papers reporting microfluidic sperm selection using the keywords: microfluidic sperm selection, self-motility, non-motile sperm selection, boundary following, rheotaxis, chemotaxis, and thermotaxis. Papers published before March 31, 2023 were selected.

OUTCOMES: Our results show that most studies have used motility-based properties for sperm selection. However, microfluidic platforms are ripe for making use of other properties such as chemotaxis and especially rheotaxis. We have identified that low throughput is one of the major hurdles to current microfluidic sperm selection chips, which can be solved via parallelization.

CONCLUSION: Future work needs to be performed on numerical simulation of the microfluidics chip prior to fabrication as well as relevant clinical assessment after the selection procedure. This would require a close collaboration and understanding among engineers, biologists, and medical professionals. It is interesting that in spite of two decades of microfluidics sperm selection, numerical simulation and clinical studies are lagging behind. It is expected that microfluidic sperm selection platforms will play a major role in the development of fully integrated start-to-finish assisted reproductive technology systems.

Atherosclerosis is the main killer in the west and therefore a major health challenge today. Total serum cholesterol and lipoprotein concentrations, used as clinical markers, fail to predict the majority of cases, especially between the risk scale extremes, due to the high complexity in lipoprotein structure and composition. In particular, low-density lipoprotein (LDL) plays a key role in atherosclerosis development, with LDL size being a parameter considered for determining the risk for cardiovascular diseases. Determining LDL size and structural parameters is challenging to address experimentally under physiological-like conditions. This article describes the biochemistry and ultrastructure of normolipidemic and hypertriglyceridemic LDL fractions and subfractions using small-angle X-ray scattering. Our results conclude that LDL particles of hypertriglyceridemic compared to healthy individuals 1) have lower LDL core melting temperature, 2) have lower cholesteryl ester ordering in their core, 3) are smaller, rounder and more spherical below melting temperature, and 4) their protein-containing shell is thinner above melting temperature.

Hemoglobin measurement is a commonly ordered laboratory test for various medical needs including trauma, treatments, surgery, screening for anemia, and regular health check-ups. While invasive hemoglobin measurement is accurate and sensitive, they are time-consuming, expensive, and painful. This has led to the popularity of the point-of-care devices, such as HemoCue. However, the point-of care testing still requires blood and causes pain for the patient. The emerging field of non-invasive hemoglobin measurement devices offers pain-free, time-efficient, affordable, and user-friendly alternatives. In the conducted study the performance and limitations of non-invasive hemoglobin devices are assessed through a literature study and pilot study on two market-available devices: BG20 and Rad-67. The BG20, measuring multiple parameters, lacks specific studies on its non-invasive hemoglobin measurement, whereas the Rad-67, focusing on fewer parameters, has been more thoroughly investigated. The conducted literature study found that their key parameter, hemoglobin, still require improvement for clinical use. Additionally, patient exclusions were often due to failures in measuring non-invasive hemoglobin. The conducted experimental pilot study, involving 15 healthy volunteers for comparison of BG20 and Rad-67 against capillary blood tests using HemoCue, supported the literature findings. Bland-Altman analysis indicated that both devices had limits of agreement too broad for clinical application, though Rad-67 showed promising bias (0.1 g/dl) compared to BG20’s larger bias (1.94 g/dl). Pearson correlation analysis revealed a moderate correlation between Rad-67 and HemoCue, but only a weak correlation for BG20 and HemoCue. In conclusion, current non-invasive hemoglobin testing technology requires further development to meet clinical standards.

The adsorption of components from black tea and of purified tea polyphenols onto a whole unstimulated salivary pellicle-like protein layer, formed in vitro on hydroxyapatite discs, was studied by in situ ellipsometry. It was found that components from black tea and the purified polyphenols epicatechin-3-gallate (ECG), epigallocatechin-3-gallate (EGCG) and theaflavin readily adsorbed onto the pellicle. Further investigations showed that under the experimental conditions of this study, no black tea- or purified polyphenol-modified pellicles were eluted by either phosphate buffer or sodium dodecyl sulphate rinses. Therefore, black tea and its polyphenol components are indicated to have a profound effect on in vitro pellicle modification. Similar effects were observed for tannic acid.

The hydrophobic interaction between two methane molecules in salt-free and high salt-containing solutions and the structure in such solutions have been investigated using an atomistic model solved by Monte Carlo simulations. Monovalent salt representing NaCl and divalent salt with the same nonelectrostatic properties as the monovalent salt have been used to examine the influence of the valence of the salt species. In salt-free solution the effective interaction between the two methane molecules displayed a global minimum at close contact of the two methane molecules and a solvent-separated secondary minimum. In 3 and 5 M monovalent salt solution, the potential of mean force became slightly more attractive and in a 3 M divalent salt solution the attraction became considerably stronger. The structure of the aqueous solutions was determined by radial distribution functions and angular probability functions. The distortion of the native water structure was increased with ion valence. The increase of the hydrophobic attraction was associated with (i) a breakdown of the tetrahedral structure formed by neighboring water molecules and of the hydrogen bonds between them and (i) the concomitant increase of the solution density.