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In vitro systems like those used for in vitro embryo production are invaluable for our understanding of embryogenesis and the processes that regulate it. However, extensive research has also highlighted that in vitro produced embryos negatively differ from their in vivo counterparts in various ways. Not surprisingly, there is ~20% decrease in pregnancy success from pregnancies established using in vitro produced embryos. Therefore, much research has relied on attempting to produce a better in vitro embryo that more closely resembles their in vivo counterparts. Our laboratory has investigated this by supplementing a cytokine, interleukin-6 (IL6), during in vitro embryo culture. My dissertation work expands upon those initial efforts by answering more detailed questions related to the biological role of IL6 during cattle embryogenesis. In the work presented herein, IL6 supplementation during in vitro culture was able to transform the transcriptome of resulting conceptuses post embryo transfer. The transcriptome of these conceptuses included an abundance of genes associated with survival. Indeed, we witnessed IL6-treated conceptuses resulted in a 20% increased survival rate and were longer than their non-treated counterparts. In the second research project, we employed CRISPR-Cas9 genome editing technology to understand the embryo phenotype after part of the IL6 receptor responsible for signal transduction, interleukin-6 signal transducer (IL6ST), is disrupted. We discovered that IL6ST is required for development before the blastocyst stage. In addition, IL6ST disrupted blastocysts, presumed to contain wildtype, presented with severe, abnormal morphology. Not only did this group of embryos have decreased ICM and TE cell numbers, but they also had an increased occurrence of cells within the TE region that were negative for its traditional marker, CDX2. This suggests IL6ST is likely involved in a pathway responsible for determining cell fate identity at the blastocyst stage. Collectively, IL6 in cooperation with IL6ST, is a key controller of embryogenesis in cattle. / Doctor of Philosophy / There are major events that an embryo must successfully advance from to continue development to form into an organism capable of survival after birth. Over 30% of pregnancies in cattle and humans will fail within the first 30 days of gestation. This time period coincides with several key developmental events that ultimately modify the morphology of the growing embryo. Our laboratory primarily focuses on embryo development around the blastocyst stage. If an embryo advances to this stage, it has a greater likelihood of maintaining viability. Therefore, my dissertation research has focused on early embryonic development from the time of first cleavage (~day 2 of gestation) through embryo elongation (~day 15 of gestation), which encompasses the blastocyst stage. Within this time frame, I have been investigating embryonic effects after supplementation of a protein, interleukin-6 (IL6). Previously, our laboratory has identified IL6 to cause favorable impacts on the developing embryo, but its mode of action was unknown. Therefore, my dissertation research has investigated the mechanistic actions of IL6, and its beta receptor subunit, interleukin-6 signal transducer (IL6ST). In my first research project, we discovered that supplementing IL6 during in vitro embryo culture resulted in increased embryo elongation and survival. In my second research project, we found IL6ST is an absolute requirement for embryo survival to the blastocyst stage. Together, these results indicate IL6 is a very important protein needed for sustained pregnancy viability.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2010. / Härtill 5 uppsatser.

Interleukin-10 ist ein Paradebeispiel eines immunhemmenden Zytokins. Es konnte nachgewiesen werden, dass eine Reihe von Tumoren Interleukin-10 produziert, um einer Antitumor-Immunantwort zu entgehen. Viele Studien haben sich mit dem Einfluss von Interleukin-10 auf die antigenpräsentierenden Fähigkeiten der Dendritischen Zellen beschäftigt. Es gibt eindeutige Hinweise, dass der Effekt von tumorproduziertem Interleukin-10 nicht nur in einer hemmenden Wirkung auf die Ausreifung Dendritischer Zellen besteht, sondern dass Interleukin-10 zu einer Reduktion der Anzahl an Dendritischen Zellen führen kann. Ziel dieser Arbeit ist es daher, den Mechanismus für eine solche depletierende Wirkung auf die Dendritischen Zellen zu analysieren. Hierzu wurden die Effekte von Interleukin-10 auf die frühe Differenzierung von Dendritischen Zellen aus Monozyten untersucht. Die Zugabe von Interleukin-10 zu einem Differenzierungscocktail aus Interleukin-4 und Granulozyten/Makrophagen-Kolonie-stimulierendem-Faktor führt zu einer nachhaltigen Hemmung des Differenzierungsprozesses von Monozyten zu Dendritischen Zellen. Bereits 48h nach Beginn der Zellkultur konnte mit Hilfe von cDNA-Microarray-Analysen gezeigt werden, dass Interleukin-10 nicht nur einen Differenzierungs-hemmenden Effekt ausübt, sondern auch die Entstehung aberranter Zellphänotypen bewirkt. In weiteren Experimenten konnte gezeigt werden, dass die Effekte des Interleukin-10 in der frühen Differenzierungsphase weitgehend irreversibel sind. Zusammenfassend können die Ergebnisse zur Erklärung beitragen, wie es bei Patienten mit Tumoren unter dem Einfluss von Interleukin-10 zu einer Reduktion der absoluten Zahl Dendritischer Zellen kommen kann.:1. Verzeichnis der Abkürzungen 5 2. Einleitung 7 Fragestellung 11 3. Material und Methoden 12 3.1 Verwendete Materialien 12 3.1.1 Reagenzien für Zellseparation und Zellkultur 12 3.1.2 Stimulatoren 12 3.1.3 Reagenzien für FACS-Analysen 12 3.1.4 Molekularbiologische Reagenzien und Puffer 13 3.1.4.1 RNA-Isolierung 13 3.1.4.2 RT-PCR 13 3.1.4.3 cDNA Microarrays 14 3.2 Zellen und Kulturbedingungen 15 3.2.1 Isolierung von Monozyten aus dem peripheren Blut 15 3.2.2 Ausreifung von Monozyten zu Dendritischen Zellen 16 3.3 Durchflusszytometrie 17 3.4 RNA Isolierung 18 3.4.1 Prinzip und Protokoll der RNA-Isolierung 18 3.4.2 Quantifizierung der RNA und Bestimmung der Reinheit 19 3.4.2.1 Photometrische Bestimmung der RNA-Konzentration 19 3.4.2.2 Agarosegelelektrophorese 20 3.4.2.3 RNA-Fällung 21 3.5 cDNA Expressions-Ansätze 22 3.5.1 mRNA-Amplifikation 22 3.5.2 Membranmarkierung 23 3.5.2.1 Prinzip des cDNA-Array 23 3.5.2.2 Probensynthese 24 3.5.2.3 Aufreinigung 25 3.5.2.4 Hybridisierung der Nylonmembran 25 3.5.2.5 Auswertung der Ergebnisse 27 3.6 Statistische Auswertung 27 4. Ergebnisse 28 4.1 Differenzierung von Monozyten zu unreifen Dendritischen Zellen unter dem Einfluss von IL-4 und GM-CSF 28 4.2 Ermittlung der wirksamen hemmenden Konzentration von IL-10 29 4.3 Einfluss von IL-10 während der Differenzierung von Monozyten zu unreifen Dendritischen Zellen 30 4.3.1 Oberflächenexpression nach 7 Tagen 30 4.3.2 Oberflächenexpression nach 2 Tagen 32 4.3.3 Oberflächenexpression nach 24 Stunden und 48 Stunden für die Chemokinrezeptoren CCR1 und CCR7 33 4.4 Ausreifung von unreifen Dendritischen Zellen zu reifen Dendritischen Zellen unter dem Einfluss von KLH, TNF-α und GM-CSF 36 4.5 Kann eine adäquate Ausreifung die hemmenden Effekte von IL-10 überwinden? 37 4.6 Einfluss von IL-10 während der Ausreifung Dendritischer Zellen 38 4.7 Genexpressionsmuster in der frühen Phase der Differenzierung Dendritischer Zellen 40 5. Diskussion 49 5.1 Hintergrund 49 5.2 Einfluss von IL-10 auf die Differenzierung von Monozyten zu unreifen Dendritischen Zellen 51 5.3 Einfluss von IL-10 während der Ausreifung Dendritischer Zellen 53 5.4 Genexpression 55 5.4.1 Übersicht 55 5.4.2 Regulation von CCR1 und CCR2 durch IL-10 56 5.4.3 Regulation von IL-1ß und IL-1R1 durch IL-10 58 5.4.4 Regulation von S100A8 und S100A9 durch IL-10 59 5.5 Biologische Bedeutung 61 6. Zusammenfassung 63 7. Literaturverzeichnis 67

Background Nutritional treatment in the intensive care unit (ICU) has evolved from meeting nutritional requirements to manipulating patient outcome. Pharmaconutrition, referring to nutrients that are applied for their pharmacological properties, forms part of the standard nutritional care plan. The most abundant amino acid in the body, glutamine, is also the most-researched pharmaconutrient. It is an independent predictor of mortality in ICU patients, at both deficient and very high levels. Glutamine supplementation is recommended in the ICU setting for its proven outcome benefits. However, recent data showed that glutamine supplementation increases mortality risk in certain patient groups. Moreover, it suggested that not all ICU patients are glutamine deficient. Therefore, the main aim of this study was to investigate the plasma glutamine levels of adult ICU patients, on admission to the ICU. In addition, to elucidate the profile of ICU patients that can be expected to present with a glutamine deficiency or excess, with regards to gender, diagnosis and inflammatory markers. Methods In this observational, cross-sectional study, 60 mixed ICU adult patients admitted to two hospitals in the North West province were included in the study group. Blood sampling was conducted within 24 hours following ICU admission, to determine plasma glutamine, interleukin (IL)-6 and C-reactive protein (CRP) levels. Plasma glutamine levels were compared with those of a control group of healthy individuals, matched by age, race, and gender. Gender-related differences in plasma glutamine levels were investigated, as well as differences between patients with various medical conditions. The relationship between plasma glutamine levels and IL-6 or CRP was examined. Additionally, a CRP concentration cut-off point at which glutamine becomes deficient was determined by means of a receiver operating characteristic (ROC) curve. Results and discussion Intensive care unit patients had significantly lower plasma glutamine levels than healthy individuals on day one of ICU admission (p < 0.0001). However, only 38.3% (n = 23) had deficient plasma glutamine levels (< 420 μmol/L), while 6.7% (n = 4) presented with supra-normal levels (> 930 μmol/L). No significant difference could be detected between the plasma glutamine levels of male and female ICU patients (p = 0.116). Likewise, levels between diagnosis categories were also not significantly different (p = 0.325). There was a significant inverse association between plasma glutamine levels and CRP concentrations (r = -0.44, p < 0.05), and a trend towards an inverse association with IL-6 (r = - 0.23, p = 0.08). A CRP cut-off value of 95.5 mg/L was determined, above which plasma glutamine values became deficient; however, more research is needed to confirm this result. Conclusion and recommendations This research therefore showed that ICU patients, when compared with healthy individuals, had lower plasma glutamine levels on day one of admission to the ICU. However, not all were glutamine deficient, as the majority had normal and some presented with supra-normal plasma glutamine levels. An individualised approach should therefore be followed in identifying candidates for glutamine supplementation. The patients‟ condition alone may not be sufficient to predict glutamine status, but an association between plasma glutamine levels and CRP was firmly established, as well as a cut- off CRP-value above which glutamine can be expected to become deficient, which could be of use in this regard. / MSc (Dietetics), North-West University, Potchefstroom Campus, 2015

Background Nutritional treatment in the intensive care unit (ICU) has evolved from meeting nutritional requirements to manipulating patient outcome. Pharmaconutrition, referring to nutrients that are applied for their pharmacological properties, forms part of the standard nutritional care plan. The most abundant amino acid in the body, glutamine, is also the most-researched pharmaconutrient. It is an independent predictor of mortality in ICU patients, at both deficient and very high levels. Glutamine supplementation is recommended in the ICU setting for its proven outcome benefits. However, recent data showed that glutamine supplementation increases mortality risk in certain patient groups. Moreover, it suggested that not all ICU patients are glutamine deficient. Therefore, the main aim of this study was to investigate the plasma glutamine levels of adult ICU patients, on admission to the ICU. In addition, to elucidate the profile of ICU patients that can be expected to present with a glutamine deficiency or excess, with regards to gender, diagnosis and inflammatory markers. Methods In this observational, cross-sectional study, 60 mixed ICU adult patients admitted to two hospitals in the North West province were included in the study group. Blood sampling was conducted within 24 hours following ICU admission, to determine plasma glutamine, interleukin (IL)-6 and C-reactive protein (CRP) levels. Plasma glutamine levels were compared with those of a control group of healthy individuals, matched by age, race, and gender. Gender-related differences in plasma glutamine levels were investigated, as well as differences between patients with various medical conditions. The relationship between plasma glutamine levels and IL-6 or CRP was examined. Additionally, a CRP concentration cut-off point at which glutamine becomes deficient was determined by means of a receiver operating characteristic (ROC) curve. Results and discussion Intensive care unit patients had significantly lower plasma glutamine levels than healthy individuals on day one of ICU admission (p < 0.0001). However, only 38.3% (n = 23) had deficient plasma glutamine levels (< 420 μmol/L), while 6.7% (n = 4) presented with supra-normal levels (> 930 μmol/L). No significant difference could be detected between the plasma glutamine levels of male and female ICU patients (p = 0.116). Likewise, levels between diagnosis categories were also not significantly different (p = 0.325). There was a significant inverse association between plasma glutamine levels and CRP concentrations (r = -0.44, p < 0.05), and a trend towards an inverse association with IL-6 (r = - 0.23, p = 0.08). A CRP cut-off value of 95.5 mg/L was determined, above which plasma glutamine values became deficient; however, more research is needed to confirm this result. Conclusion and recommendations This research therefore showed that ICU patients, when compared with healthy individuals, had lower plasma glutamine levels on day one of admission to the ICU. However, not all were glutamine deficient, as the majority had normal and some presented with supra-normal plasma glutamine levels. An individualised approach should therefore be followed in identifying candidates for glutamine supplementation. The patients‟ condition alone may not be sufficient to predict glutamine status, but an association between plasma glutamine levels and CRP was firmly established, as well as a cut- off CRP-value above which glutamine can be expected to become deficient, which could be of use in this regard. / MSc (Dietetics), North-West University, Potchefstroom Campus, 2015

BACKGROUND:Migraine headache is one of the most common neurological disorders, but the pathophysiology contributing to migraine is poorly understood. Intracranial interleukin-6 (IL-6) levels have been shown to be elevated during migraine attacks, suggesting that this cytokine may facilitate pain signaling from the meninges and contribute to the development of headache.METHODS:Cutaneous allodynia was measured in rats following stimulation of the dura with IL-6 alone or in combination with the MEK inhibitor, U0126. The number of action potentials and latency to the first action potential peak in response to a ramp current stimulus as well as current threshold were measured in retrogradely-labeled dural afferents using patch-clamp electrophysiology. These recordings were performed in the presence of IL-6 alone or in combination with U0126. Association between ERK1 and Nav1.7 following IL-6 treatment was also measured by co-immunoprecipitation.RESULTS:Here we report that in awake animals, direct application of IL-6 to the dura produced dose-dependent facial and hindpaw allodynia. The MEK inhibitor U0126 blocked IL-6-induced allodynia indicating that IL-6 produced this behavioral effect through the MAP kinase pathway. In trigeminal neurons retrogradely labeled from the dura, IL-6 application decreased the current threshold for action potential firing. In response to a ramp current stimulus, cells treated with IL-6 showed an increase in the numbers of action potentials and a decrease in latency to the first spike, an effect consistent with phosphorylation of the sodium channel Nav1.7. Pretreatment with U0126 reversed hyperexcitability following IL-6 treatment. Moreover, co-immunoprecipitation experiments demonstrated an increased association between ERK1 and Nav1.7 following IL-6 treatment.CONCLUSIONS:Our results indicate that IL-6 enhances the excitability of dural afferents likely via ERK-mediated modulation of Nav1.7 and these responses contribute to migraine-related pain behavior in vivo. These data provide a cellular mechanism by which IL-6 in the meninges causes sensitization of dural afferents therefore contributing to the pathogenesis of migraine headache.