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Bilirubinwerte im Säuglingsalter und deren Assoziation zum Körpergewicht, zu den Konzentrationen von Eisenstoffwechselparametern und Steroidhormonen
No full textIt is assumed that bilirubin is hormonally regulated and influences weight development by preventing weight gain. However, studies in healthy infants are limited. The present study established reference values for bilirubin and investigated whether bilirubin levels are significantly associated with body weight, levels of ferritin and transferrin as well as steroid hormone levels in a study population of three- and six-month-old healthy infants. Data from a total of 411 study visits from the LIFE Child study (Leipzig, Germany) were analyzed. Associations were examined using linear regression analyses. Besides laboratory parameters, anthropometric data were gathered. We found statistically significant associations between body weight and bilirubin levels. In girls, we observed additional associations between bilirubin levels and both ferritin and transferrin concentrations at three months of age. At six months, steroid hormone levels were significantly associated with concentrations of total and indirect bilirubin, with effects differing by sex. Our study thus confirms associations already reported from animal studies and studies in adult populations. Furthermore, we showed that these associations already exist in the first year of life, are influenced by sex and age and, further, depend on the bilirubin type. Our results provide reference values for bilirubin and assist, therefore, in interpreting bilirubin levels in infancy
Adhesion G protein-coupled receptors in adipocytes and their functional relevance
No full textG protein-coupled receptors (GPCRs) are expressed ubiquitously throughout the human body
and are involved in the regulation of almost all physiological functions. Their role in
adipogenesis and mature adipocytes has been thoroughly studied. Expression of a high number
of GPCRs in adipose tissue has been shown, among them many orphan receptors. The continued
increase in obesity worldwide creates an urgent need to understand the physiological role of
those orphan receptors in adipose tissue functions and to identify targets for new
pharmacological therapies (Chooi et al., 2019b). Members of the adhesion GPCR (aGPCR)
class belong to these orphan receptors and their function in adipocytes and adipose tissue is
very poorly understood. Only recently, more evidence emerged showing aGPCR expression in
adipocytes and their regulating functions (Al Hasan et al., 2020; Nie et al., 2012b). The
objective of this study was to provide a comprehensive overview of all members of the aGPCR
class regarding their expression and function in fat tissue.
Analysis of publicly available RNA-Seq data from mouse visceral adipose tissue and
qPCR analysis of mouse visceral and subcutaneous adipose tissue detected mRNA expression
of several aGPCRs. Receptor expression differed in subcutaneous fat compared to visceral fat.
qPCR analysis of mouse stromal vascular fraction and mouse adipocytes from visceral adipose
tissue revealed mRNA expression of almost all aGPCRs. When comparing diet-induced obese
mice with lean mice, upregulation as well as downregulation of aGPCRs was found in many
cases, both in subcutaneous and visceral adipose tissue. Analysing RNA-Seq data of
subcutaneous adipose tissue of children from the Leipzig Adipose Tissue Childhood Cohort,
significant regulation of mRNA expression of aGPCRs was observed in obese individuals
compared to lean children. These findings point towards a regulating role of aGPCRs in obesity
in mice and humans.
qPCR analysis of aGPCR expression in 3T3-L1 cells revealed expression of several
aGPCRs. During differentiation, expression of aGPCRs showed different patterns. Knockdown
of aGPCRs via siRNA and staining cells with Oil Red O, which specifically stains lipids and
triglycerides, revealed reduced total lipid accumulation for six aGPCRs. siRNA-mediated
knockdown of these aGPCRs also resulted in lowered mRNA expression of the differentiation
marker Pparγ, suggesting a regulatory impairment of lipogenesis after receptor knockdown.
Analysis of fatty acid composition showed a trend towards longer fatty acyl chains after
knockdown of two aGPCRs.
Endogenous GPR64/ADGRG2 activation using the Stachel-derived peptide pGPR64 resulted
in increased cAMP and, subsequently, lipolysis in 3T3-L1 cells. This was also observed in
primary mouse adipocytes. Stimulation of endogenous GPR64/ADGRG2 in 3T3-L1 cells
caused reduced adiponectin secretion and less insulin-dependent glucose uptake. These results
show that aGPCRs regulate preadipocyte differentiation as well as adipocyte function,
highlighting their regulatory role in adipose tissue and yielding them interesting targets to
modulate adipocyte function.
3T3-L1 cells are a popular model for studying adipocyte differentiation and function. A
common method to study specific proteins is knockdown and subsequent analysis of cell
properties. While studying the function of GPR64/ADGRG2 and GPR126/ADGRG6 in 3T3-
L1 cells, knockdown of genes via siRNA presented itself as challenging as the efficiency could
be low due to differentiation into adipocytes (Suchý et al., 2020). Therefore, cell editing using
the CRISPR/Cas9 system might prove more beneficial. However, plasmid transfection in 3T3-
L1 is no choice to deliver Cas9 into the cells. Therefore, using cells overexpressing Cas9 to
generate gene knockouts promises to be a more efficient method to study genes of interest and
has become more popular in recent years (Cong et al., 2013).
Hence, the objective of this study was to create CRISPR/Cas9-generated receptor
knockouts of Gpr64/Adgrg2 and Gpr126/Adgrg6 in 3T3-L1 cells and study the ability of
receptor-depleted cells to differentiate into adipocytes. Using commercially available 3T3-L1
cells overexpressing Cas9 (Cas9 cells), cells depleted of GPR64/ADGRG2 and
GPR126/ADGRG6 were created by inserting premature stop codons into the genome, 64ko
cells and 126ko cells. As a control, 3T3-L1 cells were transfected with scrambled guide RNA,
ctrl cells. However, data revealed impaired adipocyte function not only in knockout cells but
also in control cells, indicating that continuous Cas9 expression itself changed 3T3-L1 cell
behavior. Thus, further research focused on characterizing 3T3-L1 cells expressing Cas9
compared to untreated 3T3-L1 cells.
Comparing differentiation rates of Cas9 cells to wild-type 3T3-L1 cells (wt cells), Cas9
cells showed reduced differentiation, suggesting that Cas9 expression itself already impaired
3T3-L1 cell function, expression of Pparγ and Cebpα were not affected though. Stimulation
with forskolin and isoprenaline also revealed diminished cAMP signaling in Cas9 cells
compared to wt cells, showing no changes regarding adiponectin secretion or lipolysis. Further
qPCR analysis of Cas9 cells revealed changes in mRNA expression of adrenergic receptors,
phosphodiesterase 3, insulin receptor, and hormone-sensitive lipase in Cas9 cells compared to
wt cells, showing altered adipocyte function in cells continuously expressing Cas9. Finally, wt
3T3-L1 cells were transfected with Cas9 protein. This transient presence of Cas9 in 3T3-L1
cells did not impair cell differentiation, as there were no changes in lipid accumulation
compared to wt cells. This could serve as an alternative to using Cas9 cells without disrupting
wt cell function.:Introduction ............................................................................................................................... 2
Obesity and adipose tissue ................................................................................................................ 2
G protein-coupled receptors and their physiological relevance .................................................... 3
GPCR function in adipocytes – Gs and Gi signaling ..................................................................... 5
GPCR function in adipocytes – Gq signaling ................................................................................. 5
Adhesion G protein-coupled receptors ............................................................................................ 7
Reliability of model cell lines and transferability to animal models in obesity research ............ 8
Objectives and Aims .......................................................................................................................... 9
Publications ............................................................................................................................. 10
The repertoire of Adhesion G protein-coupled receptors in adipocytes and their functional
relevance ........................................................................................................................................... 10
Evaluating the feasibility of Cas9 overexpression in 3T3-L1 cells for generation of genetic
knock-out adipocyte cell lines ......................................................................................................... 24
Summary of work .................................................................................................................... 40
Bibliography ............................................................................................................................ 45
Supplementary information .................................................................................................... 52
Declaration of own contribution ............................................................................................. 81
Erklärung über die eigenständige Abfassung der Arbeit ...................................................... 88
Curriculum Vitae ..................................................................................................................... 89
List of scientific publications and presentations .................................................................... 91
Publications ...................................................................................................................................... 91
Posters .............................................................................................................................................. 91
Danksagung ............................................................................................................................. 9
Untersuchung magnetischer Oszillatoren und zweier magnetisch gekoppelter Fadenpendel
No full textIn dieser Masterarbeit werden schwingungsfähige Systeme (Oszillatoren) mithilfe von Magneten aufgebaut. Die Eigenfrequenzen werden zunächst auf Grundlage des Dipolmodells theoretisch berechnet und anschließend im Experiment gemessen. Dabei lassen sich Analogien zu klassischen mechanischen Oszillatoren ziehen. Darüber hinaus zeigt sich bei zwei magnetisch gekoppelten Fadenpendeln, dass es möglich ist, eine negative Kopplungskonstante einzustellen.:1. Motivation
2. Theoretische Grundlagen
3. Technische Daten
4. Magnetfeld- und Kraftmessung
5. Magnetschwinger zwischen zwei Magneten
6. Magnetschwinger auf einer geneigten Ebene
7. Zwei magnetisch gekoppelte Fadenpendel
8. Fazit und Ausblick
A. Anhang
Literatur
SelbstständigkeitserklärungIn this master's thesis, oscillatory systems (oscillators) are constructed using magnets. The natural frequencies are first calculated theoretically on the basis of the dipole model and then measured experimentally. Analogies to classical mechanical oscillators can be drawn here. Furthermore, two magnetically coupled string pendulums show that it is possible to set a negative coupling constant.:1. Motivation
2. Theoretische Grundlagen
3. Technische Daten
4. Magnetfeld- und Kraftmessung
5. Magnetschwinger zwischen zwei Magneten
6. Magnetschwinger auf einer geneigten Ebene
7. Zwei magnetisch gekoppelte Fadenpendel
8. Fazit und Ausblick
A. Anhang
Literatur
Selbstständigkeitserklärun
Gut-Brain Interactions after Bariatric Surgery: on the Relationship of Gut Hormones and Working Memory
No full textAdipositas ist eine chronische Erkrankung mit hohem Gesundheitsrisiko und steigender Prävalenz. Die bariatrische Chirurgie gilt als wirksamste Langzeittherapie für anhaltende Gewichtsreduktion, weist jedoch eine hohe interindividuelle Variabilität der Ergebnisse auf. Mechanistisch werden der Gewichtsreduktion u. a. tiefgreifende postoperative Veränderungen von Stoffwechsel, Appetit und Sättigung zugeschrieben – insbesondere eine veränderte Ausschüttung hormoneller Signale der Darm-Hirn-Achse (GLP-1, PYY, Ghrelin). Parallel sind bei Adipositas Beeinträchtigungen exekutiver Funktionen, vor allem des Arbeitsgedächtnisses, beschrieben, für die nach bariatrischer Chirurgie teilweise Verbesserungen berichtet werden. Unklar ist, inwieweit Darm-Hirn-Signale diese kognitiven Veränderungen und einen nachhaltig erfolgreichen Gewichtsverlauf mitbestimmen.
In der vorliegenden Arbeit wurde dieser Zusammenhang konzeptionell untersucht, indem langfristig erfolgreiche und weniger erfolgreiche Operierte gegenübergestellt und die postprandiale Sekretion von Magen-Darm-Hormonen sowie Leistungen des Arbeitsgedächtnisses erfasst wurden. Insgesamt zeigte sich, dass günstigere Langzeitergebnisse mit einer appetitzügelnden hormonellen Antwort und besseren Leistungen in komplexeren Arbeitsgedächtnisaufgaben einhergingen. Explorativ deutete sich an, dass insbesondere die Dynamik des Hormons Ghrelin den Zusammenhang zwischen Gewichtsreduktion und Kognition vermitteln könnte.
Die Ergebnisse ordnen bariatrische Behandlungserfolge in ein neurobiologisches Rahmenmodell ein, in dem dopaminerge Prozesse eine Brückenrolle zwischen Energiehaushalt, Essverhalten und kognitiver Kontrolle spielen könnten. Daraus ergeben sich Perspektiven für personalisierte Nachsorge und Interventionen, die Darm-Hirn-Signale gezielt modulieren. Angesichts des post-hoc-Studiendesigns und der begrenzten Stichprobengröße werden größere und prospektive Studien empfohlen – idealerweise mit zerebraler Bildgebung und unter Einbezug weiterer potenzieller Mediatoren wie Entzündungsstatus, Vagusnerv, Mikrobiom und Gallensäuren.:1 Introduction 3
1.1 Obesity – a Global Challenge 3
1.2 Therapy Success and Variability of Bariatric Surgery 3
1.3 Mechanisms behind Weight Loss after Bariatric Surgery 4
1.4 Gut-Brain Communication after Bariatric Surgery 5
1.5 The Role of Cognitive Functions in Obesity and after Bariatric Surgery 9
1.6 Research Aims 10
1.7 A Central Method for Assessing Working Memory 11
2 Original Publication 13
3 Summary 29
3.1 Introduction and Research Question 29
3.2 Methods 30
3.3 Results and Discussion 31
3.4 Implications 33
4 Bibliography 35
5 Appendix 51
5.1 List of Abbreviations 51
5.2 Academic Contribution 53
5.3 Declaration of Authenticity 54
5.4 Curriculum Vitae 55
5.5 Publications 56
5.6 Acknowledgments 5