Charles University University of Porto Faculty of Pharmacy in Hradec Králové Faculty of Pharmacy Department of Pharmacology and Toxicology Department of Biological Sciences Student: Karolína Zubatá Supervisor: doc. PharmDr. Martina Čečková, Ph.D. Consultants: Susana Rocha, Ph.D., prof. Alice Santos-Silva, Ph.D. Title of diploma thesis: Oxidative stress biomarkers of the erythrocyte in the newborn - a follow-up study Increased levels of oxidative stress (OS) have been described in healthy, full-term newborns as a consequence of the drastic changes introduced by birth and by the exposure to extrauterine environment. Our intention was to examine the OS levels in red blood cells (RBCs) of neonates and to further understand the changes that the newborn organism undergoes with its newly- acquired autonomy as this knowledge is limited and there are no reference values. Umbilical cord blood samples were collected from a small population of newborns (n = 8) and several hematological and biochemistry parameters were evaluated. Our experimental data consist of OS biomarkers measurements performed in different fractions of blood (RBC membrane, total RBCs and plasma): membrane bound hemoglobin (MBH), lipid peroxidation (LPO), quantification of catalase (CAT) and glutathione peroxidase (GPx) activities,...

The ploidy level of diploid and induced triploid tench, Tinca tinca, was verified using flow cytometry to determine relative DNA content of 4',6-diamidino-2-phenylindole (DAPI)-stained erythrocyte nuclei. The C-value (haploid nuclear DNA content; pgDNA.nucleus-1) of these same individuals was determined by means of Feulgen image analysis densitometry, in comparison to the chicken standard (Gallus gallus domesticus; 1.25 pgDNA.nucleus-1, P < 0.05), using three different approaches. Highly similar mean C-values were obtained, thus confirming the possibility of using tench blood as standard in European pond aquaculture for ploidy and DNA content determination in fishes. Feulgen image analysis densitometry (FIAD), flow cytometry (FC) and confocal laser scanning microscopy (CLSM) were used to study the relationship between the DNA content (pgDNA.nucleus-1), nuclear area (?m2), nuclear volume (?m3) and 3-D structure of erythrocyte nuclei in a series of fish ploidy level models: diploid tench (Tinca tinca) (2n), Cuban gar (Atractosteus tristoechus) (2n), triploid tench (3n), evolutionary tetraploid sterlet (Acipenser ruthenus) and stellate sturgeons (A. stellatus) (4n), evolutionary octaploid Siberian sturgeon (A. baerii) and Russian sturgeon (A. gueldenstaedtii) (8n), spontaneous triploid Siberian and Russian sturgeons exhibiting dodecaploidy (12n), evolutionary 12n shortnose sturgeon (A. brevirostrum), and experimentally obtained sturgeon hybrids that were tetraploid, hexaploid (6n), heptaploid (7n), octaploid (8n), decaploid (10n), dodecaploid (12n) and/or tetradecaploid (14n). Standards used for FIA were blood smears of chicken (2.5 pgDNA.nucleus-1), diploid and induced triploid tench (2.04 and 3.1 pgDNA.nucleus-1, respectively). All ploidy levels were first verified by means of FC. Increase in ploidy was accompanied by growth of the nucleus and an increase in the number of flattened ellipsoid nuclei with increased transverse diameter. The volume (Vvoxel) of erythrocyte nuclei, as the sum of voxels calculated from live cells, seems more accurate than volume (Vaxis) calculated from measuring the major and minor axis, especially at higher and odd ploidy levels. Data of absolute and relative DNA content were in agreement with previously published reports. Species of the same ploidy level, however differing in their DNA content, exhibited a similar mean erythrocyte nuclear area, as could be demonstrated on A. ruthenusand and A. stellatus (19.27 and 19.79 ?m2 with a respective mean DNA content of 3.72 and 4.68 pgDNA.nucleus-1) and volume as could be demonstrated on a A. ruthenus and hybrid of A. ruthenus and H. huso(48.3 and 48.9 ?m3 with a respective mean DNA content of 3.74 and 3.10 pg DNA.nucleus-1). Similar relationship was found for the ploidy 6n, 8n, 10n, 12n. The 0.46-1.58 pgDNA increments in DNA content of erythrocytes thus had no effect on their nuclear area/volume. With increasing ploidy level, the DNA concent ration (pgDNA per 1 ?m3 of erythrocyte nuclear volume) as well as surface-to-volume ratio was found not to increase linearly. Nuclear DNA content appeared to be more condensed with an increase of the ploidy level. Observed results deduce properties of whole cell and particularly of the nuclei in series of ploidy levels fishes, adding conformations of nucleotypic hypothesis in context of cell/nuclear size and genome size relationships, as well as taxonomic position of sturgeons.

Syre transporteras i kroppen från lungorna till vävnader bundet till hemoglobin i erytrocyter. Specifikt i hemoglobin är det järnatomen som binder syret. För att det ska bildas erytrocyter är det nödvändigt med järn, folsyra och vitamin B12. Brist på dessa leder till anemi.  Anemi drabbar ca 1/3 av världens befolkning. Det definieras som minskning av friska erytrocyter och leder till att kroppens vävnader inte får tillräckligt med syre. Den vanligaste formen av anemi orsakas av järnbrist. Järnbrist förekommer i två former: absolut eller funktionell. Absolut järnbrist är centralt i detta arbete och innebär låga eller tomma järnförråd som kan leda till järnbristanemi. Denna sker vid bland annat ökat behov, minskat upptag av järn från födan, malabsorption eller kroniska blödningar. Ungefär 1,2 miljarder människor i världen idag har järnbristanemi. De vanligaste symtomen är huvudvärk, blek hud, trötthet och dyspné. Under graviditeten så tredubblas järnbehovet för att kunna försörja moderns ökande blodvolym och erytrocyter, tillväxten av fostret samt placentan och för att kompensera för blodförlusten vid förlossningen. Har man låga eller tomma järnförråd innan graviditeten så riskerar man järnbristanemi. Järnbristanemi hos gravida ökar risken för prematur födsel, låg födelsevikt och missfall. Risken för mödradödlighet har en direkt korrelation med järnbrisanemins svårighetsgrad. Om järnbristanemi lämnas obehandlad så har det nyfödda barnet en ökad risk att själv drabbas av järnbristanemi och det kan även ha en negativ påverkan på barnets kognitiva utveckling. Vid behandling av järnbristanemi ges i första hand oralt järn men vid svårare fall så ges intravenöst järn. Syftet med studien var att jämföra effektivitet och säkerhet av intravenös och oral järn-administration vid järnbristanemi i samband med graviditet och postpartum hos kvinnor. Metoden som används går ut på flera sökningar på Pubmed där sex kliniska studier som passar frågeställningen valdes ut för arbetet. Resultatet från alla dessa artiklar pekar på att intravenös behandling av järnbristanemi är mer effektiv på att öka Hb värden och järndepåer med ökad följsamhet och mindre antal biverkningar. Av det testade preparaten gav järnkarboxymaltos mest lovande resultat. Trots att arbetet hade flera begränsningar som kunde ha förbättrats genom att använda fler homogena kliniska studier så gynnade resultaten från alla sex studier den intravenösa behandlingen över den orala behandlingen. Dock borde alla gravida kvinnor ta oralt järntillskott tidigt i förebyggande syfte, redan under den första trimestern för att undvika utveckling av järnbristanemi. Intravenöst järn bör användas som sista utväg vid svårare typer av anemi, malabsorption samt för att öka följsamheten. / Oxygen is transported in the body from the lungs to tissues while bound to hemoglobin in erythrocytes. Specifically in hemoglobin it is the iron atom that binds the oxygen. Synthesis of erythrocytes place iron, folic acid and vitamin B12. A lack of these compounds leads to anemia. Anemia affects about 1/3 of the world's population. It is defined as lower-than-normal levels of healthy erythrocytes and deprives the body's tissues of oxygen. The most common form of anemia is caused by iron deficiency. Iron deficiency occurs in two forms, absolute or functional iron deficiency. Absolute iron deficiency is central for this study. Absolute iron deficiency implies low or empty iron storages which can cause iron deficiency anemia. This occurs in the event of, among other factors, increased demand for iron, reduced iron content in the food, malabsorption or chronic bleedings. Approximately 1.2 billion people have iron deficiency anemia worldwide. The most common side effects are headache, paleness, fatigue and dyspnea. During pregnancy the need for iron triples to be able to support the mother's increase of blood plasma, erythrocytes, the growth of the fetus and the placenta and to compensate for the blood loss during childbirth. If you have low or empty iron stores before pregnancy, you are at risk for iron deficiency anemia. Iron deficiency anemia in pregnant women increases the risk of prematurity, low birth weight and miscarriage. The risk of maternal mortality has a direct correlation with the severity of iron deficiency anemia. If iron deficiency anemia is left untreated, the newborn has an increased risk of suffering from iron deficiency anemia and also a negative impact on the cognitive development. When treating iron deficiency anemia, oral iron is the primarily treatment but in more severe cases intravenous iron is given. The objective of this study was to compare the efficacy and safety of intravenous and oral iron treatment for iron deficiency anemia in pregnancy and postpartum women. The method used consists of several searches on Pubmed where six clinical studies that fit the aim of this report were chosen. The results from all these articles indicate that intravenous treatment of iron deficiency anemia is more effective at increasing Hb values ​​and iron storages with increased compliance and fewer side effects. The iron preparation that was most promising was ferric carboxymaltose. Although this study had several limitations that could have been improved by using more homogeneous clinical studies, the results from all six studies favored the intravenous treatment over the oral treatment. However, all pregnant women should take oral iron supplements early on as a preventive measure in the first trimester to avoid developing iron deficiency anemia. Intravenous iron should be used as a last resort in more severe types of anemia, malabsorption and to increase compliance.