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How to reduce weaning and separation stress in dairy cow-calf contact systems? A comparison of a gradual process with the two-step nose flap method and an evaluation of different stress indicators
(2024) Vogt, Anina
Early separation of dairy cows and their calves shortly after birth is increasingly questioned by society, scientists and some farmers. Cow-calf contact (CCC) systems, which allow extended contact between a calf and their dam or a foster cow, are an alternative to this practice with many benefits for the animals’ welfare, but the weaning and separation process remains a major challenge in CCC systems. The aim of this thesis was therefore to compare the behavioral and physiological stress responses of dairy cows and their 3-months-old calves during weaning and separation either via two-step weaning using nose flaps (NF, 2 wks full-time contact while calves wore nose flaps, 1 wk fence-line contact before total separation) or via a gradual reduction of cow-calf contact time (GR, 1 wk half day contact, 1 wk morning contact, 1 wk fence-line contact before total separation, n=18 cow-calf pairs per method). As a secondary aim different indicators for weaning and separation stress were evaluated.
The behavioural responses of 36 cow-cow pairs were directly observed in the cow area and via video monitoring in the calf area, which also included the selection gate where calves could switch between areas. Locomotor play levels and lying times of calves, as well as lying and rumination time of cows, were automatically recorded with sensors. Blood and fecal samples were collected for analysis of fecal cortisol metabolite concentrations, relative telomere length and immune responses. Milk yield was continuously recorded, and calves were weighed weekly.
For the cows, no significant differences were found between the two separation methods in any of the used behavioral or physiological indicators. However, both methods led to an increase in vocalizations and searching behaviour compared to baseline at several time points, as well as to a transient increase in physiological stress markers, indicating that both methods provoked stress for the cows. For calves, the abrupt milk loss through the nose flap seemed to compromise adaptation of the gastrointestinal tract and led to low weight gains, reduced play behaviour and an increase in inflammatory blood markers. Furthermore, calves with nose flaps exhibited a marked decrease in lying and play behaviour, along with a high number of unsuccessful suckling attempts, which pointed towards a negative emotional state in NF calves. In contrast, GR calves showed higher weight gains, a lower decrease in lying bouts as well as a lower decrease in locomotor play levels during weaning compared to baseline, indicating a better adaption of the gastrointestinal tract to the dietary change as well as a likely less compromised affective state. In addition, the nose flap had caused pressure marks and injuries at the nasal septum of calves after the 14 days of usage. Results from our video analyses suggested that the calves needed to wear the nose flap for at least 4 days in order to effectively reduce suckling motivation, while there seems to be no benefit of using nose flaps longer than this time. However, it could not be determined whether nasal septum injuries were already present in our NF calves after the 4 days of usage.
Given the overall beneficial effect of the gradual weaning and separation method for the calves and the missing difference in cows, the GR method seems overall favorable to reduce weaning and separation stress for cow-calf pairs compared to the NF method. Nonetheless, the GR method also caused considerable stress for the animals and recommendations for refinement are given in the thesis, highlighting, among others, the need to truly reduce the milk intake of calves and to focus on loosing established routines rather than just contact time itself.
With regard to the stress indicators, it became evident that a low frequency of vocalizations during weaning should not be taken as evidence of low stress levels of animals, if not backed by further indicators. Relative telomere length as sampled in the present thesis was not a valid indicator of cumulative separation stress, while calves’ fecal cortisol metabolite concentrations were found to be a generally valid stress indicator for (unweaned) dairy calves, but unsuitable for comparing pre- and post-weaning states.
In conclusion, this thesis provides valuable new insights to improve the weaning and separation process in CCC systems, that directly support practical applications as well as future research on this topic.
Research on CuxTi1-xO2 Thin Films Acting as Buffer Layers for VO₂-Based Smart Window Applications (Raw data)
(2025-03-26) Hao Lu
This folder contains the raw experimental data of Cu-doped TiO₂ thin films, which will be used for the writing and publication of my scientific paper. It also includes some optical data of VO₂ films grown on CuTiO₂ buffer layers.
About experiment:
The CuxTi1-xO2 layers were deposited by radio-freqency (RF) sputtering on quartz substrates (Suprasil). We used a 4-inch target of ceramics TiO2 at a distance of 4 inches from the substrate. In order to alloy the TiO2 thin films with Cu, several pure Cu lines were mounted onto the target manually. This combination allowed us to achieve CuTiO2 alloy with various Ti:Cu ratios and, thus, to deposit CuxTi1-xO2 thin films. All thin films were sputtered to obtain a thickness from 100 to 200 nm at 200°C to 400°C heater temperature. A mixture of Ar and O2 with a pressure of 3.4×10−3 mbar were used to generate the plasma. The O2 gas flux was varied between 0–3 sccm at a fixed Ar flux of 31 sccm. Anatase and rutile thin films were prepared by ion beam sputtering at room temperature and 560°C, respectively. VO2 thin films were prepared via RF sputtering using a 4-inch metallic vanadium target. The deposition temperature of the VO2 films was controlled within the range of 300°C to 400°C. The RF plasma was generated using a mixture of Ar and O2 gases at a pressure of 3.4×10−3 mbar. The gas flux ratio was set at 1.1 sccm for O2 and 31 sccm for Ar. All VO2 thin films had a thickness of 50 nm.
The layer thicknesses and densities were analyzed with X-ray Reflection (XRR). The film structure was analyzed with Grazing-Incidence X-Ray Diffraction (GIXRD). In this mode of XRD, the X-rays are incident on the sample at a grazing angle, meaning they skim along the surface rather than striking it directly perpendicularly. Thus, this shallow angle enables an enhanced sensitivity to surface structures. Both, XRR and XRD, were performed using a Rigaku SmartLab diffractometer that operates a 9 kW rotating Cu anode. X-ray Photoelectron Spectroscopy (XPS) was employed for the comprehensive analysis of both the elemental composition within the film and the valence states inherent to each individual element. We used a PHI VersaProbe II spectrometer with a monochromated Al Kα (1486.6 eV) X-ray anode directed at 45° towards the surface normal. Charge compensation was achieved using a combination of an electron gun and an Ar+ ion gun. The samples were exposed to a focused Ar+ beam at an acceleration voltage of 1 kV to remove adsorbed impurities. Raman spectroscopy with 515 nm laser excitation and a spectral resolution of 1.5 cm−1 (Renishaw inVia Raman microscope system) was used for phase identification of the CuxTi1-xO2 and VO2 thin films, providing complementary confirmation to the findings obtained by XRD. A Zeiss-Merlin scanning electron microscope (SEM) equipped with an InLens detector was used for investigating the film morphology. Optical transmittance measurements were conducted using a PerkinElmer Lambda 900 UV–Vis–NIR spectrometer. For temperature-dependent measurements, a resistively heated sample holder, controlled by a Eurotherm heating controller and integrated with a Peltier cooling device, was used to achieve precise heating and cooling rates. A PT100 sensor was employed to monitor the sample's actual temperature. Optical simulations were conducted using the Essential MacLeod software, based on optical constants obtained from spectroscopic ellipsometry for all constituent layers, TiO2, VO2 and CuxTi1-xO2, respectively.
Effekt von BDNF auf die Angiogenese im humanen Kokultursystem aus Endothelzellen und osteoporotischen Osteoblasten
(2025) Holtkamp, Katharina Ines Carmen
Die Bildung und Stabilisierung von Blutgefäßen ist für die Frakturheilung von entscheidender Bedeutung. Im Herz konnte gezeigt werden, dass das Signalmolekül brain-derived neurotrophic factor (BDNF) die Angiogenese fördert. Vor diesem Hintergrund stellte sich die Frage, ob durch eine Applikation von BDNF die Bildung von Blutgefäßen stimuliert wird und ob die Osteoblasten osteoporotischer Spender hierbei regulierend wirken. Angelehnt an die gestellte Hypothese konnte kürzlich aufgezeigt werden, dass mesenchymale Stammzellen (MSC) und Osteoblasten bei Osteoporose phänotypische Veränderungen im Vergleich zu knochengesunden Zellen aufweisen. Zur Beantwortung der Frage wurden humane Osteoblasten und Endothelzellen im Kokultursystem untersucht. MSC wurden aus humaner Spongiosa isoliert, die bei der Endoprothetik als Restmaterial anfiel. Es wurden MSC von n = 5 knochengesunden und n = 5 osteoporotischen Spendern verwendet. Für das Kokultursystem wurden die MSC 10 Tage osteogen differenziert. Anschließend wurden immortalisierte humane dermale mikrovaskuläre Endothelzellen (HMEC), in einem Verhältnis von 1:4, sowie BDNF (20 ng/mL Medium) hinzugefügt. Nach 3 und 5 Tagen wurde der von-Willebrand-Faktor (vWF) im Zellkulturmedium mittels ELISA bestimmt. Die Zellen wurden ebenfalls nach 3 und 5 Tagen geerntet und zur Bestimmung der relativen alkalischen Phosphatase (ALP) Aktivität verwendet. Abschließend erfolgte eine statistische Analyse und graphische Darstellung. Die Konzentration des sezernierten vWF im Zellkulturmedium stieg von Tag 3 zu Tag 5 signifikant in Kokulturen mit humanen Osteoblasten von osteoporotischen Spendern nach Zugabe von BDNF an. In Kokulturen knochengesunder Osteoblasten konnte ein vergleichbarer Anstieg der vWF Konzentration auch ohne die zusätzliche Applikation von BDNF gemessen werden. Die Differenzierung und Aktivität der Osteoblasten wurde durch die relative Aktivität der ALP ermittelt. Es waren keine signifikanten Unterschiede in der ALP Aktivität in den Kokulturen knochengesunder und osteoporotische Osteoblasten, sowie nach Applikation von BDNF zu verzeichnen.
Die Ergebnisse zeigen, dass Osteoblasten osteoporotischer Spender nach Zugabe von BDNF keinen negativen Einfluss auf die Bildung des vWF und somit auf die Angiogenese ausüben. Diese stimulierende Wirkung von BDNF ist in Kokulturen knochengesunder Osteoblasten nicht notwendig. BDNF zeigte im Kokultursystem mit humanen Endothelzellen keinen Einfluss auf die ALP Aktivität, die als Zeichen für die Aktivität der Osteoblasten verwendet wurde. Zusammenfassend zeigte sich durch BDNF eine positive Beeinflussung der osteoporotischen Kokultur in Bezug auf die Angiogenese, womit ein zukünftiger Ansatz im Umgang mit Osteoporose aufgezeigt werden konnte.
Factor XII in community-acquired pneumonia - Sex specific differences and correlation between clinical parameters
(2025) Ehrlich, Kristin
Pneumonia accounts for one of the ten main causes of death worldwide with highest incident rates in small children and elderly people. Generally, men are more susceptible to an infection and show elevated rates of hospitalization.
The role of FXII as a coagulation factor has been highly questioned as people deficient in FXII do not show any kind of bleeding disorder and with the discovery of a TF- dependent coagulation pathway in vivo, FXII seemed to be dispensable for hemostasis. Therefore, current studies focus on alternative roles of FXII. A growing number of evidence supports the theory that FXII is directly and indirectly associated with inflammatory processes and thrombosis and thereby is playing an important role for innate host defense against infection. Multiple sources showed that FXII does not only lead to fibrin formation but also to production and release of proinflammatory cytokines and stimulation of neutrophils.
Although previous studies highlighted the role of FXII and its downstream products during ARDS, a common complication of pulmonary infection, corresponding data for FXII in the context of pneumonia is missing. The present study investigated levels of FXII, FXIIa and HK in plasma of 140 CAP patients and 60 sex- and age-matched healthy donors by performing analysis of data collected via western blot and ELISA. The results were further analyzed to determine sex-specific differences and correlations with CRP levels, CRB-65-score and mortality. Additionally, FXII levels in BALF of 20 CAP patients and 10 donors was measured by western blot and ELISA. Finally, immunohistochemical stainings for FXII and pro-SP-C, CD-68 and vWF in human lung tissue of 3 CAP patients were performed.
Previous studies showed that levels of FXII and its inhibitor decline simultaneously during infection, hypothesizing a progressive consumption of both. In contrast to that I found elevated levels of FXIIa-C1INH-complexes in plasma of CAP patients, while no parallel decrease of FXII levels was observed. That could be a sign of either inhibited degradation of FXII or stimulation of production and secretion of FXII. Multiple studies suggest neutrophils as a source of FXII and show a proinflammatory influence of estrogen on neutrophil function.
In line with these findings the present work shows sex specific differences with regard to FXII and HK levels in CAP patients. Female CAP patients showed higher amounts of FXII than sex-matched donors, while HK levels in women suffering from CAP were decreased in comparison to sex-matched donors. As estradiol is known to stimulate FXII production and studies have shown an association between estradiol levels and FXII levels in plasma, these observations let me speculate that changes of FXII plasma levels may be influenced by alterations of estradiol levels in the healthy elderly population as well as in the context of pulmonary infection. Epidemiological data show that male individuals are more prone to infectious diseases than female individuals. These sex- specific differences in the susceptibility to but also the outcome of pulmonary infection are hypothesized to originate from sex-hormone-dependent but also sex-hormone- independent variations of innate and adaptive immune responses. The results presented add further evidence to sex specific differences in the context of pulmonary infection and emphasize the need to support further research on that matter.
Moreover, the sex-specific differences in FXII and HK levels let me speculate about the role of FXII and its downstream products with regard to immunothrombosis as women are more prone to thrombosis and elevated levels of estrogens are related to an increased risk of thromboembolic events. Currently contradictory results have been published, leaving the question of a rather protective or a harmful role of FXII and its downstream products in the context of inflammation and related coagulopathy unanswered. That is why further studies regarding this question need to be performed. Although I would have expected more correlations of FXII and its downstream products with clinical parameters, only a negative correlation of CRP levels and FXII levels in women with CAP, as well as a corresponding positive correlation with CRP levels and FXIIa-C1INH complexes in female CAP patients was detected. Furthermore, a negative correlation of HK levels and the CRB-65 score in female CAP patients was found. The weak correlations to CRP and CRB-65 score imply a supplementary, rather than a substitutive role of FXII, FXIIa and HK in the prediction of disease outcome of CAP. Finally, elevated levels of FXII in BALF of CAP patients were shown, indicating local production or accumulation of plasma-derived FXII in CAP lungs.
Via immunolocalization of FXII to endothelial cells a binding of FXII to endothelial cells has been proposed in this study. While FXII-uPAR-associated signalling seems to have a rather protective role in the context of inflammation the consequent activation of the KKS by FXIIa can result in extensive vascular inflammation.
Immunolocalization of FXII to the surface of macrophages implies a binding of FXII to macrophages with a probable consequent activation of FXII into FXIIa leading to a secretion of pro-inflammatory cytokines and fatal disease outcome.
Moreover, FXII was immunolocalized to ATII cells which, together with endothelial cells, play an important role in maintaining a functioning capillary-alveolar barrier, which is essential for host defense. In this study a possible association of FXII and the regulation of ATII cell function is proposed.
All in all, the work presented shows altered levels of FXII, FXIIa and HK during CAP with sex-specific differences, indicating that FXII plays an important role in the context of pulmonary inflammation and associated coagulopathy. Not only the role of FXII as an effector of host defense but especially sex-specific effects of FXII should be investigated further in the future. Only then we will be able to draw conclusions on individual disease outcome and can develop personalized therapeutic strategies.
Functional characterization of the membrane-depolarizing toxin TisB in Escherichia coli
(2025-01) Leinberger, Florian Hartmut
Bacteria are frequently exposed to environmental stressors that threaten their survival. This leads to the activation of stress response systems that adjust gene expression to maintain cellular in- tegrity. However, if these systems are inadequate, bacteria can form persister cells. These are a dormant subpopulation with reduced metabolic activity and increased antibiotic tolerance. Unlike other forms of bacterial dormancy, such as endospores, persister cells are morphologically similar to active cells but are in a transient state of reduced activity. This survival strategy allows bacteria to withstand adverse conditions that can lead to relapse of infection and the spread of antibiotic resistance.
This work examines the functionality of TisB, a toxin of the type I toxin-antitoxin system tisB/istR-1 in Escherichia coli (E. coli), and its role in maintaining dormancy through specific physiological mechanisms. The research suggests that insertion of TisB into the inner membrane leads to membrane depolarization and ATP depletion, which can be considered as key triggers for the dormant state associated with antibiotic tolerance. The results presented illustrate the influence of TisB on persister cell physiology and its role in bacterial survival under antibiotic stress. An important aspect of this work is the use of a moderate expression system for TisB, which allows a controlled investigation of its effects without causing excessive toxicity. This allows for an exam- ination of the relationship between TisB-induced dormancy and protein aggregation. The results show that TisB-dependent protein aggregation influences the duration of dormancy during antibi- otic exposure. In addition, amino acids critical for TisB functionality were identified. This provided insight into the structural elements that are essential for its activity. These findings contribute to a deeper understanding of TisB and highlight the importance of specific amino acids in maintaining its functionality within the membrane.
In summary, the results presented here deepen our understanding of the tisB/istR-1 system and its role in bacterial persistence. By elucidating the mechanisms by which TisB influences persistence, protein aggregation and energy content, this study provides a foundation of knowledge that can serve as a starting point for developing strategies to curb bacterial persistence and improve the efficacy of antibiotics.