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Der Einfluss einer Parabenexposition auf die Entstehung von Übergewicht und allergischem Asthma bronchiale – eine Analyse im experimentellen Mausmodell
No full textIn den letzten Jahrzehnten ist die Prävalenz für Übergewicht und Asthma bronchiale
kontinuierlich angestiegen. Neben einer genetischen Veranlagung spielen
Umweltchemikalien eine große Rolle. Die Parabene n-Butylparaben (nBuP) und Ethylparaben (EtP) stehen im Verdacht, den Hormonhaushalt im Körper zu beeinflussen und erste epidemiologische Studien konnten zeigen, dass diese mit einer Krankheitsentstehung im Kindesalter wie z. B. der atopischen Dermatitis durch mütterliche Exposition während der Schwangerschaft assoziiert sein können [1]. In der vorliegenden Arbeit wurde in einem generationsübergreifenden Mausmodell der Einfluss einer Exposition mit nBuP bzw. EtP auf die Gewichtsentwicklung und das allergische Asthma analysiert. Eine nBuP-Behandlung der Mäuse während Gravidität und Laktationsperiode führte zu einem erhöhten Gewicht, der selbige Einsatz von EtP zu einem verstärkten Asthma-Phänotyp, beides ausschließlich in weiblichen Nachkommen. Als mögliche zugrundeliegende Mechanismen konnten für das
veränderte Körpergewicht eine Hypermethylierung des für die neuronale Sättigungsregulation hauptverantwortliche POMC-Gens (POMC – Proopiomelanocortin) und in Bezug auf den Asthma-Phänotyp eine veränderte Expression von Asthma-relevanten Genen identifiziert werden. Im adulten Versuchsmodell beeinflusste eine direkte Exposition der Mäuse mit nBuP
bzw. EtP das Körpergewicht nicht. Interessanterweise hatte nBuP einen Asthma
reduzierenden Effekt, der durch EtP in der adulten Maus nicht beobachtet werden konnte. Zusammenfassend konnte gezeigt werden, dass eine Paraben-Exposition während Gravidität und Laktationsperiode in Abhängigkeit der Expositionssubstanz das Körpergewicht und den Asthma-Phänotyp der weiblichen Nachkommen negativ beeinflussen kann. Durch den ubiquitären Einsatz von Parabenen kann eine geringfügige Exposition des Fötus bereits während der Schwangerschaft nicht vermieden werden. Durch die Verwendung von parabenfreien Kosmetika und Lebensmitteln kann die Belastung aber minimiert und das Risiko für allergische Erkrankungen und Übergewicht im Kindesalter nicht zusätzlich erhöht werden
Am Rande des Bekannten: Extrem elektronenreiche (Di)carboranylphosphane
No full textDie Synthese der ersten B9-verbundenen Carboranylphosphane
(B9-Phos) mit zwei Carboranyleinheiten
sowie der Zugang zu B9-Phos-Liganden mit
sperrigen elektronen-schiebenden Substituenten, die bisher
als unerreichbar galten, werden vorgestellt. Die
elektrochemischen Eigenschaften der B9-Phos-Liganden
wurden untersucht und die Fähigkeit der Mesityl-Derivate,
stabilisierte Phosphoniumylradikalkationen zu bilden,
wurde gezeigt. Die B9-Phos-Liganden weisen einen
extrem elektronenschiebenden Charakter auf, der den
von Alkylphosphanen und allgemein verwendeten Nheterocyclischen
Carbenen übertrifft. Dies wird durch
ihre sehr kleinen elektronischen Tolman-Parameter
(TEP) sowie durch extrem niedrige P Se-Kopplungskonstanten
belegt. Kegelwinkel und verdeckten Volumina
belegen die hohe sterische Hinderung, die von den
(Di)carboranylphosphanen ausgeht. Die Dicarboranylphosphan-
AuI-Komplexe zeigen im Vergleich zu den
Monocarboranylphosphan-Analoga eine bessere katalytische
Leistung bei der Hydroaminierung von Alkinen
Robert Mugabe and Patronage Politics in Zimbabwe, 1980-2017
No full textThis Doctor of Philosophy (PhD) dissertation analyses Zimbabwe’s former, and now deceased, long serving President Robert Mugabe’s patronage politics of 37 years, from 1980 to 2017. A lot is known, but less understood about the complexity of Mugabe’s longevity in power - largely because, most literature has concentrated on the hero/villain binary existentiality of his patronage politics, whilst ignoring his self-construction and the people’s perspectives about him. The quadruple complex, as a totality as this dissertation’s point of departure, aids the analysis on how Mugabe managed to consolidate and sustain himself in power. The focus of this dissertation is the prosopographical analysis of Mugabe’s hero-villain-self-constructed complexity, and what people said about him, through his neopatrimonialism political practices, and shows how the domestic and international institutions and actors played out when Mugabe was consolidating his power.:TABLE OF CONTENTS
ACKNOWLEDGEMENTS II
ABSTRACT III
TABLE OF CONTENTS IV
LIST OF ACRONYMS VII
CHARPTER ONE
1 INTRODUCTION 1
1.1 State of the Art Literature Review 1
1.2 Conceptualising patronage, patrimonialism and Neopatrimonialism 1
1.2.1 Evolution of neopatrimonialism in Africa 3
1.3 Robert Mugabe’s systematic neo-patrimonialism strategies 7
1.4 Robert Mugabe’s neo-patrimonialism within the state functioning 16
1.5 Case studies from African countries 21
1.5.1 Gabon’s Omar Bongo 21
1.5.2 Sudan’s Omar al Bashir 24
1.6 Research Question 28
1.7 Problem Statement 28
1.8 Justification 29
1.9 Research Methodology 29
1.9.1 Prosopography 29
1.9.2 Research design 33
1.9.3 Research Method 33
1.9.4 Data Generation Method 34
1.9.5 Method of Data Analysis 38
1.9.6 Challenges in the field 38
1.10 Structure of the dissertation 39
CHAPTER TWO
2 ROBERT MUGABE’S SYSTEMATIC NEOPATRIMONIALISM STRATEGIES 40
2.1 Mugabe and the security sector patronage politics 40
2.2 Capacities for violence and access to rents 40
2.2.1 The Army 41
2.2.2 The Police 45
2.2.3 The Intelligence 47
2.3 The Informal Security Sector 49
2.4 Personalised appointments of security commanders 54
2.5 Crises and threats to Robert Mugabe’s rule and the role of the security sector in quashing dissent 58
2.6 Shortcomings and the Breakdown of Robert Mugabe’s Neopatrimonialism 74
2.7 Some conclusions on the security sector 78
2.8 Robert Mugabe’s Utilisation of the Youths 80
2.9 Non-Military Business Complex 90
CHAPTER THREE
3 ROBERT MUGABE’s COMPLEX EXISTENTIALITY 96
3.1 Self-construction 96
3.1.1 Mugabe as a deity 97
3.1.2 Patriarch/administrator of the Zimbabwean estate (father of the nation) 98
3.1.3 Mugabe the Martyr 99
3.1.4 Nativist (son of the soil) 101
3.1.5 Mugabe the victim 103
3.2 Mugabe’s self-construction and his patronage for power sustenance: an analysis 104
3.3 Perspectives of Zimbabwean citizens on Robert Mugabe 107
3.3.1 The Optimists 109
3.3.2 The Radicals 118
3.3.3 The Cautionists 127
CHAPTER FOUR
4 ROBERT MUGABE’s INTERNATIONAL RELATIONS AND PATRONAGE POLITICS OF SURVIVAL 135
4.1 Robert Mugabe’s patronage politics with neighbouring countries 136
4.1.1 South Africa and Mozambique 137
4.1.2 Zambia and Botswana 142
4.2 Robert Mugabe and his regional patronage politics with regional bodies -SADC, IGAD, AU 146
4.2.1 Mugabe’s SADC Patronage Politics-Mozambique War 146
4.2.2 SADC Patronage Politics-The DRC War 147
4.2.3 IGAD Patronage Politics- Great Lakes Relations 148
4.2.4 Patronage politics at African Union /continental level 149
4.3 Robert Mugabe’s patronage politics with the international community 152
4.3.1 Robert Mugabe’s patronage politics with China and Western Countries Relationships 152
4.3.2 Mugabe’s patronage politics with rhetoric regards the UK, EU and America 153
4.4 Failure of international patronage, the sanctions agenda and return to domestic patronage. 165
CHAPTER FIVE
5 RESEARCH FINDINGS AND CONCLUSIONS 175
5.1 Introduction 175
5.2 Mugabe is not only a binary hero-villain leader but a much more complicated political actor 177
5.3 Institutions with the capacity for violence as enablers for regime consolidation and sustenance 178
5.4 Robert Mugabe’s power consolidation and sustenance through international relations patronage and oratory rhetoric 180
5.5 Is Zimbabwe a failed state? 182
5.6 Conflicts between parties and fragility of political environment as catalysts for regime consolidation and sustenance 183
5.7 Contribution to theory 184
5.8 Contribution to research 186
5.9 Implications for future research 187
6 REFERENCES
6.1 Literature 188
6.2 Newspapers and other online media sources 205
ERKLÄRUNG
Fight INflammation to Improve outcome after aneurysmal Subarachnoid HEmorRhage (FINISHER) trial: Study protocol for a randomized controlled trial
No full textAneurysmal subarachnoid hemorrhage (SAH) has high morbidity and mortality. While the primary injury
results from the initial bleeding cannot currently be influenced, secondary injury through vasospasm and delayed cerebral
ischemia worsens outcome and might be a target for interventions to improve outcome. To date, beside the aneurysm
treatment to prevent re-bleeding and the administration of oral nimodipine, there is no therapy available, so novel treatment
concepts are needed. Evidence suggests that inflammation contributes to delayed cerebral ischemia and poor outcome
in SAH. Some studies suggest a beneficial effect of anti-inflammatory glucocorticoids, but there are no data from
randomized controlled trials examining the efficacy of glucocorticoids. Therefore, current guidelines do not recommend
the use of glucocorticoids in SAH
Characterization of the suppressive activity of canine non-conventional TCRαβposCD4negCD8αneg double-negative T cells
No full textDiese Studie konnte erstmals zeigen, dass nicht-konventionelle TCRαβpos dn
T-Zellen des Hundes potente Suppressor-Zellen in vitro sind und einzigartige suppressive Eigen
schaften im Vergleich zu murinen und humanen dn T-Zellen aufweisen. Sie erweitert darüber hinaus
das Verständnis der konventionellen caninen CD4pos Treg. Angesichts des immuntherapeutischen
Potenzials regulatorischer T-Zellenen, z. B. bei Allergie oder Autoimmunität, ist die Identifizierung
nicht-konventioneller caniner dn T-Zell-Subpopulationen in vitro von großer Relevanz. Weitere Stu
dien sind erforderlich, um ihre Rolle in vivo zu erforschen
Metabolic effects of the body fat percentage associated locus COBLL1 (rs6738627) are associated with T2D, metabolism of glucose, lipids and adipokines
No full textIntroduction
• By 2016, obesity had burgeoned into a significant public health concern, affecting over a quarter of Europe's population. Its ramifications extend beyond mere prevalence, delving into an array of associated health risks.
• While obesity universally elevates health risks, the extent and nature of comorbidities differ among individuals. Intriguingly, the distribution of body FD holds more sway in dictating metabolic and cardiovascular threats than mere obesity levels.
• What mechanisms drive adipose tissue generation in specific depots, and how do sc and vis AT differentially contribute to obesity's pathophysiology and its associated comorbidities?
• It's clear that not all obese individuals face the same health risks. While sc, found just beneath the skin, may offer some protective effects, the accumulation of vis, which resides deep within the abdominal cavity, is unequivocally linked to the development of numerous disorders.
• The genetic landscape of obesity and FD is complex, with substantial heritability. Despite numerous genetic variants linked to BMI and body FD identified in GWAS, their explicit role in obesity and FD and how they correlate with gene expression in human AT remains a primary focus.
Aims/Questions
• How does the COBLL1 locus variant (rs6738627), linked with FD, influence genes governing AT biology?
• Does COBLL1 mRNA expression in diverse AT depots correlate with metabolic characteristics?
VI. SUMMARY
81
• What's the association of SNP rs6738627 with metabolic features and AT mRNA expression of COBLL1?
• How does Cobll1 modulate adipocyte biology in vitro?
Methods/Tasks
• Samples and clinical data were acquired from a population of patients with obese:
- Blood samples from the primary cohort (n=2860).
- paired AT samples from a subset (n=560)
• Paired AT depots intensively analyzed within an age, sex, and BMI-corresponding sub-group.
• Comprehensive genotyping and mRNA assessments were undertaken.
• Differential mRNA expressions were assessed in the paired AT samples.
• Discrepancies in COBLL1 expression between sc and vis AT were highlighted.
• Genotypic patterns of rs6738627 were contrasted in obese individuals, specifically targeting those diagnosed with T2D.
• Differences and associations with clinical data were validated in the total cohort.
• In vitro evaluations were conducted to elucidate function of Cobll1 in adipocyte biology.
Results
• The expression of COBLL1 in sc AT was higher than in vis AT.
• COBLL1 expression in adipose tissue have sexual dimorphism which was higher in sc AT compared to vis AT was more pronounced in women.
• The expression of COBLL1 was up-regulated in T2D patients compared to that of NGT patients in the vis AT, whereas in the sc AT, was down-regulated in T2D patients.
• COBLL1 expression in sc AT may a protective factor against metabolic disorder which was due to an excess of adipose tissue accumulation in individual with more than 40 kg/m2.
• BFP-locus rs6738627 identified by GWAS was significantly associated with TG, FPG, adiponectin and leptin serum levels.
• Rs6738627 was a potential candidate locus for T2D, specifically, A-allele may a defense factor against T2D.
• Cobll1 knock-down in mice adipocytes could increase TG accumulation instead of regulate adipogenesis.
• Cobll1 lower expression in mice adipocytes were not significant changes in cell morphology and structure.
VI. SUMMARY
82
• Our data did not demonstrate that COBLL1 is the causal gene for rs6738627, nevertheless, it cannot be overlooked other gene interaction with COBLL1 as a causal gene set such as GRB14 which was high correlated with COBLL1 expression in adipose tissue.
Key Considerations
• COBLL1 Expression Specificity: COBLL1 expression in AT is fat depot-specific and is related to obesity and T2D. Specifically, COBLL1 expression is higher in sc AT than in vis AT.
• Implications of Adverse FD gene expression: It's well-established that vis/sc COBLL1 expression is associated with clinical outcomes such as lipid and metabolic diseases.
• Sexual Dimorphism: COBLL1 expression in AT exhibits notable sexual dimorphisms, with the expression difference being more pronounced in females.
• Genetic Variant and Metabolic Traits: Specific genetic variants, like rs6738627, are associated with metabolic traits related to obesity and adverse body FD.
• Role of COBLL1 in Lipid Metabolism: In vitro gene functional experiments indicate that Cobll1 might play a role in lipid metabolism, especially in adipogenesis.
• Gene Interactions: A significant correlation was observed between the expression of COBLL1 and the GRB14, suggesting that these adjacent genes might share common promoters.
• Study Limitations:
-Sample Size: Even though COBLL1 expression was measured in a relatively large sample, this sample size might still limit the study's power to detect weaker correlations.
-Multiple Testing: Many of the nominally significant associations between SNPs and traits related to adverse FD or obesity might not withstand corrections for multiple testing.
-Experimental Limitations: In vitro experiments using only one cell type might not fully mimic the in vivo scenario.
-Species Differences: The physiological differences between species (mice vs. humans) must be taken into account.:CONTENTS
LIST OF ABBREVIATIONS ..... 4
I. INTRODUCTION .....10
1 Obesity: global epidemiology ...... 10
1.1. Epidemiology ..... 10
1.2. Clinical diagnosis of obesity and measurement of fat distribution ..12
1.3. Comorbidities associated with obesity and adverse body fat distribution ... 13
1.3.1 Obesity comorbidities ........13
1.3.2 Obesity management ......15
1.4. Which factors determine obesity and fat distribution? ..... 16
1.5. Adipose Tissue (AT) ...... 17
1.5.1 The endocrine function of AT ......17
1.5.2 AT distribution .......18
1.5.3 Vis vs. sc AT.......19
1.5.4 AT dysfunction in obesity ........... 21
1.5.5 Adipogenesis ...........22
1.6 Genetics of obesity ........... 24
1.6.1 Syndromic obesity ............. 24
1.6.2 Monogenic obesity (Mendelian non-syndromic obesity) ....... 25
1.6.3 Common polygenic obesity ........... 26
1.7 Genetics of fat distribution ...........30
1.8 Cordon-Bleu WH2 Repeat Protein Like 1 (COBLL1) .......35
1.8.1 The role of GWAS loci close to COBLL1 ..........35
1.8.2 Homologous genes of COBLL1 and protein function ........ 36
1.8.3 COBLL1 in obesity ........... 37
II HYPOTHESIS AND OBJECTIVES ......... 43
III MATERIALS AND METHODS ....... 44
1 Subjects and cohorts ............. 44
1.1 Leipzig Obesity BioBank ..........44
2 Methods ...... 46
2.1 Analysis of human mRNA expression ........ 46
2.2 Genotyping of single-nucleotide polymorphisms ............ 48
2.3 Cell culture and group design ............ 49
2.4 Target gene knock down (KD) ........... 51
2.5 RNA and protein extraction (cell) ..........52
2.6 Measurement of triglyceride ......... 52
TABLE OF CONTENT
3
2.7 Reverse transcription polymerase chain reaction and quantitative real-time PCR (cell) ........ 53
2.8 Western blot (WB) ............53
3 Statistical Analysis ................... 54
IV RESULTS .............55
1 COBLL1 mRNA expression and SNP rs6738627 associate with parameters of obesity and diabetes ....... 55
1.1 COBLL1 mRNA expression in AT correlates with parameters of obesity and body FD ........ 55
1.2 Genetic variant rs6738627 is associated with T2D, glucose metabolism, lipids and adipokines ....... 60
1.3 The association between rs6738627 and parameters of obesity and diabetes according to different diabetes susceptibility groups. ......... 67
1.4 Rs6738627 genotype-specific mRNA expression in AT. ........ 68
1.5 Mediation analyses .......... 69
2 Characterisation of Cobll1 effects on adipogenesis in vitro ......69
2.1 Effect of Cobll1 silencing in vitro on lipid accumulation during cell differentiation in murine adipocyte cell lines ...........69
2.2 Effects of siRNA mediated silencing of the Cobll1 on lipid accumulation in adipocytes ...... 70
2.3 Effects of siRNA mediated silencing of the Cobll1 on cell morphology ....70
2.4 Cobll1 KD in vitro .........71
2.5 Cobll1 protein expression ......72
V DISCUSSION ....... 74
1 COBLL1 expression is fat depot-specific and related to obesity and T2D ........75
2 Genetic variants in COBLL1 are associated with metabolic traits related to obesity and adverse body FD ........ 77
3 COBLL1 plays a role in lipid metabolism ........79
VI SUMMARY ................... 80
VII REFERENCES ...............83
VIII SUPPLEMENTARY DATA ................95
1. LIST OF FIGURES .................95
2. LIST OF TABLES ................. 96
3. STATEMENT OF ORIGINALITY ..............97
4. CURRICULUM VITAE .................98
5. PUBLICATIONS ......................9
The enzymatic regulation of CCL2 in murine brain after ischemic stroke
No full textStroke continues to be a relevant subject in modern medicine. In order to find new therapeutic targets for pharmacological treatments, it is essential that the pathophysiology of stroke is clarified. Ischemia results in necrosis and neuroinflammation including release of inflammatory mediators. Among the inflammatory mediators is the chemokine CCL2 (cc chemokine ligand 2). The enzymatic regulation of CCL2 is catalyzed by glutaminyl cyclase (QC), specifically its isoform (isoQC), and dipeptidylpeptidase 4 (DP4). QC/isoQC stabilize and increase biological activity, while DP4 catalyzes proteolytic degradation and decreases biological activity. This thesis has investigated the putative regulatory enzymes of CCL2 in mouse brain 72 hours after ischemic stroke by immunohistochemical labeling. The hypothesis assumed that a lack of stabilizing enzymes (QC/isoQC) would result in a lack of potent CCL2 and therefore smaller infarct size as well as an attenuated immune response. On the other hand, a lack of DP4 and consequential increase of potent CCL2 would result in larger infarct size as well as enhanced immune response. The immune response was assessed based on the quantity and signal intensity of microglia, astrocytes, and infiltrating immune cells. Ischemia lasting thirty minutes was induced using the model of filamentous middle cerebral artery occlusion (fMCAO). To investigate CCL2 distribution in stroke in regard to regulatory enzymes, different KO animals were compared: wt (wild type), CCL2 KO, QC KO, isoQC KO and DP4 KO mice. The murine brain tissue was investigated via immunohistochemical labeling with specific antibodies and digital analysis.
In short, results revealed no significant differences between wt mice and the KO mice regarding infarct size, CCL2 quantity, astrocytes and infiltrating immune cells. However, QC KO mice and DP4 KO mice presented significantly fewer reactive microglia in ischemic tissue compared to wt mice. Labeling of QC and DP4 itself revealed altered expression in ischemic tissue as well. QC-positive cells were significantly higher more numerous in ischemic compared to non-ischemic tissue. Intensity of DP4 staining was significantly lower in ischemic compared to non-ischemic tissue.
In conclusion, while CCL2 is an important part of the inflammatory response, in this model of stroke, infarct size and immune response does not appear to be influenced by CCL2 or its distribution. At present, the data suggest that infarct size remains dependent on collateral blood supply and the duration of the occlusion. However, there are new hints of DP4 and QC involvement in pathophysiology of stroke. Considering that pharmacological DP4 and QC inhibition is already either established or currently explored, these enzymes should be further investigated in the future, as they offer a possible new approach of stroke treatment.:List of Abbreviations
List of Figures
List of Tables
1 Introduction
1.1 Stroke and neuroinflammation
1.2 CCL2 and enzymatic regulation .
1.2.1 Glutaminyl cyclases
1.2.2 Dipeptidyl peptidase 4
1.3 Glial cells and peripheral immune cells
1.3.1 Microglia
1.3.2 Astrocytes
1.3.3 Peripheral immune cells
1.4 Aim of this work
2 Methods and Materials
2.1 Animals
2.2 Generation and characterization of KO mouse lines
2.3 Surgery for fMCAO
2.4 Tissue preparation
2.5 Immunohistochemical labeling
2.6 Triple immunofluorescent labeling procedures
2.7 Devices
2.8 Microscopy
2.9 Methods of Analysis
2.9.1 Bright Field Microscopy
2.9.2 Fluorescence Microscopy
2.10 Statistical analysis
3 Results
3.1 Infarct size and volume
3.2 CCL2 distribution in case of enzyme deficit
3.3 QC immunoreactivity in ischemia
3.4 Ischemia and isoQC immunoreactivity
3.5 Effects of ischemia on DP4 immunoreactivity
3.6 NFL expression in ischemic brain tissue
3.7 Effects of enzyme deficiency on microglia cells
3.8 Effects of enzyme deficiency on astrocytes
3.9 Effects of CCL2 on the migration of MHC-II+ cells
4 Discussion
4.1 Experimental considerations
4.1.1 The method of immunohistochemistry
4.1.2 Translation from animal model to humans
4.1.3 Types of ischemic stroke in rodents
4.2 Debate on the role of CCL2 in stroke
4.3 Infarct size and neuron loss
4.4 CCL2-modulating enzymes in stroke
4.4.1 QC/ isoQC may be involved in stroke
4.4.2 DP4 might facilitate a proinflammatory environment
4.5 Immune response after ischemia
4.5.1 Microglia response in ischemia may be dependent on enzyme expression
4.5.2 Reactive astrocytosis in stroke
4.5.3 MHC-II+ cells are not solely dependent on CCL2
4.6 Conclusion
5 Summary
References
Declaration of originality
Curriculum vitae
AcknowledgementsSchlaganfall ist ein relevantes Thema in der modernen Medizin. Um neue therapeutische für pharmakologische Behandlungen zu finden, ist es essenziell die Pathophysiologie des Schlaganfalls zu untersuchen. Die Ischämie führt zu Nekrose und Neuroinflammation, die wiederum die Freisetzung entzündlicher Mediatoren nach sich zieht. Hierzu zählt das Chemokin CCL2 (CC Chemokin Ligand 2). Die enzymatische Regulation von CCL2 wird durch die Glutaminylzyklase (QC), insbesondere deren Isoform (isoQC), sowie die Dipeptidylpeptidase 4 (DP4) katalysiert. QC/isoQC stabilisieren das Chemokin und erhöhen die biologische Aktivität, während DP4 den proteolytischen Abbau katalysiert und die biologische Aktivität verringert. Diese Dissertation hat die mutmaßlichen regulatorischen Enzyme von CCL2 im Mausgehirn 72 Stunden nach einem ischämischen Schlaganfall mittels immunhistochemischer Markierung untersucht. Die Hypothese ging davon aus, dass ein Mangel an stabilisierenden Enzymen (QC/isoQC) zu einer Verminderung von wirksamem CCL2 und daher zu einem kleineren Infarkt sowie einer abgeschwächten Immunantwort führt. Dem gegenüber vermuteten wir, dass ein Mangel an DP4 und die daraus resultierende Erhöhung an wirksamem CCL2 zu einem größeren Infarkt sowie einer verstärkten Immunantwort führen würde. Die Immunantwort wurde anhand der Menge und Signalintensität von Mikroglia, Astrozyten und infiltrierenden Immunzellen analysiert. Eine Ischämie des Versorgungsgebietes der A. cerebri media wurde über einen vorübergehenden Verschluss über 30 Minuten mittels Filament-Modell (fMCAO) induziert. Zur Untersuchung der Verteilung von CCL2 im Schlaganfall im Hinblick auf regulatorische Enzyme wurden verschiedene Knockout-Tiermodelle verglichen: Wildtyp (wt), CCL2 KO, QC KO, isoQC KO und DP4 KO Mäuse. Das murine Hirngewebe wurde durch immunhistochemische Markierung mit spezifischen Antikörpern und digitaler Analyse untersucht.
Kurz gesagt zeigten die Ergebnisse keine signifikanten Unterschiede zwischen wt- und KO-Mäusen bezüglich Infarktgröße, CCL2-Menge, Astrozyten und infiltrierenden Immunzellen. Allerdings zeigten QC KO- und DP4 KO-Mäuse signifikant weniger reaktive Mikroglia im ischämischen Gewebe im Vergleich zu wt-Mäusen. Die Markierung von QC und DP4 selbst zeigte ebenfalls eine veränderte Expression im ischämischen Gewebe. QC-positive Zellen waren im ischämischen Gewebe signifikant zahlreicher als im nicht-ischämischen Gewebe. Die Intensität der DP4-Färbung war im ischämischen Gewebe signifikant geringer als im nicht-ischämischen Gewebe.
Zusammenfassend lässt sich sagen, dass CCL2 zwar ein wichtiger Bestandteil der Entzündungsreaktion ist, in diesem Schlaganfallmodell jedoch weder die Infarktgröße noch die Immunantwort maßgeblich beeinflusst. Derzeit deuten Daten darauf hin, dass die Infarktgröße weiterhin von der Kollateralversorgung und der Dauer des Verschlusses abhängt. Es gibt jedoch neue Hinweise auf eine Beteiligung von DP4 und QC an der Pathophysiologie des Schlaganfalls. Da eine pharmakologische Hemmung von DP4 und QC bereits etabliert oder derzeit erforscht wird, sollten diese Enzyme in Zukunft weiter untersucht werden, da sie einen möglichen neuen Ansatz zur Behandlung des Schlaganfalls darstellen.:List of Abbreviations
List of Figures
List of Tables
1 Introduction
1.1 Stroke and neuroinflammation
1.2 CCL2 and enzymatic regulation .
1.2.1 Glutaminyl cyclases
1.2.2 Dipeptidyl peptidase 4
1.3 Glial cells and peripheral immune cells
1.3.1 Microglia
1.3.2 Astrocytes
1.3.3 Peripheral immune cells
1.4 Aim of this work
2 Methods and Materials
2.1 Animals
2.2 Generation and characterization of KO mouse lines
2.3 Surgery for fMCAO
2.4 Tissue preparation
2.5 Immunohistochemical labeling
2.6 Triple immunofluorescent labeling procedures
2.7 Devices
2.8 Microscopy
2.9 Methods of Analysis
2.9.1 Bright Field Microscopy
2.9.2 Fluorescence Microscopy
2.10 Statistical analysis
3 Results
3.1 Infarct size and volume
3.2 CCL2 distribution in case of enzyme deficit
3.3 QC immunoreactivity in ischemia
3.4 Ischemia and isoQC immunoreactivity
3.5 Effects of ischemia on DP4 immunoreactivity
3.6 NFL expression in ischemic brain tissue
3.7 Effects of enzyme deficiency on microglia cells
3.8 Effects of enzyme deficiency on astrocytes
3.9 Effects of CCL2 on the migration of MHC-II+ cells
4 Discussion
4.1 Experimental considerations
4.1.1 The method of immunohistochemistry
4.1.2 Translation from animal model to humans
4.1.3 Types of ischemic stroke in rodents
4.2 Debate on the role of CCL2 in stroke
4.3 Infarct size and neuron loss
4.4 CCL2-modulating enzymes in stroke
4.4.1 QC/ isoQC may be involved in stroke
4.4.2 DP4 might facilitate a proinflammatory environment
4.5 Immune response after ischemia
4.5.1 Microglia response in ischemia may be dependent on enzyme expression
4.5.2 Reactive astrocytosis in stroke
4.5.3 MHC-II+ cells are not solely dependent on CCL2
4.6 Conclusion
5 Summary
References
Declaration of originality
Curriculum vitae
Acknowledgement
Gewissheit: „Ich glaube, ich meine, ich denke, ich weiß“
No full text„Ich weiß, dass ich nichts weiß“, nicht einmal, ob dieses geflügelte Wort antiken Ursprungs mit Sicherheit auf den griechischen Philosophen Sokrates zurückgeführt werden kann. Dennoch vertreten wir nicht selten zu verschiedensten Themen eine sehr klare Meinung, durchaus mit Vehemenz. Aber woher kommen unsere Meinungen und unser Wissen über die Welt? Sind die modernen Wissenschaften der einzige Weg, um belastbare Gewissheiten zu erlangen oder lehrt uns die Geschichte der Wissenschaften etwas Anderes? Und wie sieht es mit jedem Einzelnen von uns aus? Wieviel Selbsteinsicht ist erreichbar? Welche Rolle spielt unsere Sprache auf dem Weg zur Erlangung von Gewissheit? Dies sind einige der Fragen, mit denen wir uns in unserer Ringvorlesung des studium universale auseinandersetzen wollen. Wir freuen uns, dass wir Referent:innen, unter anderem aus Philosophie, Natur- und Geisteswissenschaften, gewinnen konnten, die ihr Wissen mit uns teilen werden. Hierzu laden wir Sie herzlich ein, insbesondere dazu, im Anschluss an die Vorträge mit unseren Expert:innen ins Gespräch zu kommen. In Kooperation mit der Volkshochschule Leipzig laden wir zum Abschluss unserer Ringvorlesung zudem dazu ein, gemeinsam in der Gruppe lebendige demokratische Diskussionskultur auch einmal selbst zu erproben.
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Analysis of polymeric nanoparticle properties for siRNA/DNA delivery in a tumor xenograft tissue slice air–liquid interface model
No full textBackground: Classical two-dimensional (2D) cell culture as a drug or nanoparticle test
system only poorly recapitulates in vivo conditions. Animal studies are costly, ethically
controversial, and preclude large-scale testing.
Methods and Results:We established a three-dimensional (3D) tissue slice air–liquid
interface (ALI) culture model for nanoparticle testing. We developed an optimized
procedure for the reproducible generation of large sets of tissue slices from tumor
xenografts that retain their tissue architecture. When used for the analysis of nanoparticles
based on chemically modified polyethylenimines (PEIs) to deliver siRNA or
DNA, differences in transfection efficacy and cytotoxicity between nanoparticles were
observed more clearly than in 2D cell culture. While nanoparticle efficacies between
cell culture and the tissue slice model overall correlated, the tissue slice model also
identified particularly suitable candidates whose efficacy was underestimated in 2D
cell culture and had already been shown in previous in vivo studies.
Conclusion: The ex vivo 3D tissue slice ALI culture model is a powerful system that
allows the effective evaluation of biological nanoparticle efficacy and biocompatibility
in an intact tissue environment. It is comparably inexpensive, time-saving, and follows
the 3R principle, while allowing the identification of critical nanoparticle properties
and optimal candidates for in vivo applications