Aspekter på kotrafik och driftledning på gårdar med automatisk mjölkning

This thesis consists of four case studies. The objective is to find out how to get a well functioning cow traffic in an automatic milking system. Different technical and building solutions are analysed and some adjustments are proposed for the DeLaval herd management software. Case study 1: Lactation chart comparison from traditional cow traffic and Feed First™ cow traffic Case study 2: Farm A - a study of the precedence passage and the cow traffic Case study 3: Farm B – an overview of the farm and their way of working Case study 4: Key factors – how can we judge how well a VMS farm is working? Case study 1: Lactation chart comparison from traditional cow traffic and Feed First™ cow traffic In this survey data is analysed from before and after rebuilding to Feed First™ cow traffic. Lactation graphs are compared between traditional cow traffic and Feed First™ cow traffic. This case study is not included in the official version of this document. Case study 2: Farm A – a study of the precedence passage and the cow traffic Farm A has two VMS from DeLaval in a tandem solution. The farm has Feed First™ cow traffic and a new cow traffic solution which should give precedence to a selection of cows. The cow traffic as a whole on the farm has been studied and some problems have been adjusted. Some proposals are given on which cows should be in the precedence passage and some other things that ought to be adjusted on the farm concerning the cow traffic. In a comparison made between no precedence passage and the precedence passage working with the settings they had before the start of this study, only a few cows had shortened their waiting times before milking. Case study 3: Farm B– an overview of the farm and their way of working Farm B has two VMS from DeLaval in a parallel solution. The farm is functioning very well and is under expansion. The cow traffic solution used is Feed First™. A gate has been opened up and it is possible to pass it backwards. A short study has been made to find out how this influences the cow traffic. Case study 4: Key factors – how can we judge how well a VMS farm is working? In this study key factors are presented that the farmer can use as tools to judge the performance of the farm. Thus the idea is to help the farmer to estimate areas of improvement as well as areas performing well. The farmers should be able to compare their results to other farms as well as their own farm some time ago. Abbreviations: DMI, dry matter intake; FF cow traffic, Feed First™ cow traffic; mp, (milking pen)=waiting area; ms, milking station; PMR, partly mixed ratio; pp, precedence passage=(VIP lane); trad. cow traffic, traditional cow traffic; VMS, Voluntary Milking System. Explanations: cow traffic is movements of cows between different barn facilities, to perform different activities, like eating, resting, milking or using a cow brush. Semi free cow traffic, semi guided cow traffic, selective cow traffic and selectively guided cow traffic are the same Primiparous cows have given birth to one or several calves at one occasion. Multiparous cows have given birth to calves at more than one occasion.Detta examensarbete består av fyra fallstudier. Det har som mål att utröna hur man vid automatisk mjölkning kan främja en väl fungerande kotrafik. Olika tekniska och byggnadsmässiga lösningar analyseras och vissa anpassningar av DeLavals driftledningsprogram föreslås. Fallstudie 1: Laktationskurvor jämförs från traditionell kotrafik och Feed First™ kotrafik Fallstudie 2: Gård A – en studie av företrädesfil och kotrafik som helhet Fallstudie 3: Gård B – en överblick över gården och deras sätt att arbeta Fallstudie 4: Nyckelfaktorer – hur kan vi avgöra hur bra det går för en VMS-gård? Fallstudie 1: Laktationskurvor jämförs från traditionell kotrafik och Feed First™ kotrafik I den här undersökningen analyseras data före och efter ombyggnation till Feed First™ kotrafik. Laktationskurvor jämförs från traditionell kotrafik och Feed First™ kotrafik. Studien ingår ej i den officiella versionen av detta dokument. Fallstudie 2: Gård A – en studie av företrädesfilen och kotrafiken som helhet Gård A har två mjölkningsrobotar av märket VMS från DeLaval placerade efter varandra. Gården har Feed First™ kotrafik och en ny kotrafiklösning som ska ge företräde åt kor som av någon anledning har svårt att komma fram till mjölkningsrobotarna. Kotrafiken som helhet har studerats och en del brister har rättats till. Några förslag ges på vilka kor som bör få tillträde till företrädesfilen och en del övrigt som bör åtgärdas på gården gällande kotrafiken. I en jämförelse som gjordes mellan tiden utan företrädesfil och företrädesfilen med de inställningar som tillämpades före denna studie var det endast några kor som hade förkortat sina väntetider före mjölkning. Fallstudie 3: Gård B – en överblick över gården och deras sätt att arbeta Gård B har två mjölkningsrobotar av märket VMS från DeLaval. De är placerade bredvid varandra. Gården fungerar bra och är under utvidgning. Kotrafiken fungerar enligt Feed First™-principen. En grind har helt ställts upp så att den kan passeras baklänges. Ett försök har gjorts att utröna hur detta påverkar kotrafiken. Fallstudie 4: Nyckelfaktorer – hur kan vi avgöra hur bra det går för en VMS-gård? Ett förslag har tagits fram till hjälp för lantbrukare att bedöma gårdens prestanda. Avsikten är att kunna definiera förbättringsmöjligheter såväl som väl fungerande verksamhet. Lantbrukare ska kunna jämföra sina resultat både med andra gårdar och med sin egen gård för en tid sedan

Impaired dynamin 2 function leads to increased AP-1 transcriptional activity through the JNK/c-Jun pathway

Activation of AP-1 transcription factors, composed of the Jun and Fos proteins, regulates cellular fates, such as proliferation, differentiation or apoptosis. Among other stimuli, the AP-1 pathway can be initiated by extracellular ligands, such as growth factors or cytokines, which undergo internalization in complex with their receptors. Endocytosis has been implicated in the regulation of several signaling pathways; however its possible impact on AP-1 signaling remains unknown. Here we show that inhibition of dynamin 2 (Dyn2), a major regulator of endocytic internalization, strongly stimulates the AP-1 pathway. Specifically, expression of a dominant-negative Dyn2 K44A mutant increases the total levels of c-Jun, its phosphorylation on Ser63/73 and transcription of AP-1 target genes. Interestingly, DNM2 mutations implicated in human neurological disorders exhibit similar effects on AP-1 signaling. Mechanistically, Dyn2 K44A induces AP-1 by increasing phosphorylation of several receptor tyrosine kinases. Their activation is required to initiate a Src- and JNK-dependent signaling cascade converging on c-Jun and stimulating expression of AP-1 target genes. Cumulatively, our data uncover a link between the Dyn2 function and JNK signaling which leads to AP-1 induction

Raster Image Correlation Spectroscopy and Number and Brightness Analysis

The raster image correlation spectroscopy (RICS) and number and molecular brightness (N&B) methods are used to measure molecular diffusion in complex biological environments such as the cell interior, detect the formation of molecular aggregates, establish the stoichiometry of the aggregates, spatially map the number of mobile molecules, and quantify the relative fraction of molecules participating in molecular complexes. These methods are based on correlation of fluorescence intensity fluctuations from microscope images that can be measured in a conventional laser-scanning confocal microscope. In this chapter, we discuss the mathematical framework used for data analysis as well as the parameters need for data acquisition. We demonstrate the information obtainable by the N&B method using simulation in which different regions of an image have different numbers of interacting molecules. Then, using an example of two interacting proteins in the cell, we show in a real case how the RICS and N&B analyses work step by step to detect the existence of molecular complexes to quantify their properties and spatially map their interactions. We also discuss common control experiments needed to rule out instrumental artifacts and how to calibrate the microscope in terms of relative molecular brightness

Amyloid Precursor Protein Is Trafficked and Secreted via Synaptic Vesicles

A large body of evidence has implicated amyloid precursor protein (APP) and its proteolytic derivatives as key players in the physiological context of neuronal synaptogenesis and synapse maintenance, as well as in the pathology of Alzheimer's Disease (AD). Although APP processing and release are known to occur in response to neuronal stimulation, the exact mechanism by which APP reaches the neuronal surface is unclear. We now demonstrate that a small but relevant number of synaptic vesicles contain APP, which can be released during neuronal activity, and most likely represent the major exocytic pathway of APP. This novel finding leads us to propose a revised model of presynaptic APP trafficking that reconciles existing knowledge on APP with our present understanding of vesicular release and recycling

Dynamic clustering of dynamin-amphiphysin helices regulates membrane constriction and fission coupled with GTP hydrolysis

Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission

A Feedback Loop between Dynamin and Actin Recruitment during Clathrin-Mediated Endocytosis

A live-cell imaging study reveals that a positive feedback loop between dynamin and actin contributes to efficient endocytic membrane scission

Synaptic vesicle exocytosis and increased cytosolic calcium are both necessary but not sufficient for activity-dependent bulk endocytosis

This work was supported by the Medical Research Council [Grant number: G1002117] and a PhD studentship from the Biotechnology and Biological Sciences Research Council.Activity-dependent bulk endocytosis (ADBE) is the dominant synaptic vesicle (SV) endocytosis mode in central nerve terminals during intense neuronal activity. By definition, this mode is triggered by neuronal activity; however, key questions regarding its mechanism of activation remain unaddressed. To determine the basic requirements for ADBE triggering in central nerve terminals, we decoupled SV fusion events from activity-dependent calcium influx using either clostridial neurotoxins or buffering of intracellular calcium. ADBE was monitored both optically and morphologically by observing uptake of the fluid phase markers tetramethylrhodamine-dextran and horse radish peroxidase respectively. Ablation of SV fusion with tetanus toxin resulted in the arrest of ADBE, but had no effect on other calcium-dependent events such as activity-dependent dynamin I dephosphorylation, indicating that SV exocytosis is necessary for triggering. Furthermore, the calcium chelator EGTA abolished ADBE while leaving SV exocytosis intact, demonstrating that ADBE is triggered by intracellular free calcium increases outside the active zone. Activity-dependent dynamin I dephosphorylation was also arrested in EGTA-treated neurons, consistent with its proposed role in triggering ADBE. Thus SV fusion and increased cytoplasmic free calcium are both necessary but not sufficient individually to trigger ADBE. This article is protected by copyright. All rights reserved.Publisher PDFPeer reviewe

Molecular mechanisms controlling dynamin recruitment to sites of endocytosis by SH3-domain containing proteins

Endocytosis is a general cellular mechanism, which regulates a number of important events, including recycling of synaptic vesicles. Clathrin-mediated endocytosis is the best-characterized endocytic pathway in the cell. This internalization process involves the coat protein clathrin, the adaptor protein AP2 and a number of accessory proteins. All endocytic events involve a fission step, when the membrane is severed. Fission during clathrin-mediated endocytosis is regulated by the large GTPase dynamin. Dynamin self-assembles into spirals on lipid membranes and is believed to functions as a mechanochemical enzyme, severing the membrane by force generated by a GTP hydrolysis-dependent conformational change in the spiral structure. Clathrin-mediated endocytosis is the main pathway to recycle synaptic vesicles after exocytosis in the nerve terminal. Synaptic vesicle recycling is essential for sustained neurotransmission. It occurs in the periactive zone of nerve terminals. Several endocytic proteins are known to migrate from the synaptic vesicle cluster, where they reside at rest, to the periactive zone upon stimulation and Ca2+ entry. Dynamin is dependent on interactions with SH3-domain containing proteins to localize to sites of endocytis in non-neuronal cells. In the nerve terminal, dynamin interacts with a number of SH3-domain containing proteins, but the exact roles of these interactions are not known. In this thesis, we investigate how the dynamin-binding proteins intersectin, endophilin and syndapin regulate the recruitment of dynamin to the periactive zone and the function of dynamin during fission to recycle synaptic vesicles. We show that dynamin co-localizes with intersectin and endophilin in the synaptic vesicle cluster at rest and at clathrin-coated pits in the periactive zone in stimulated nerve terminals. Intersectin is important to regulate the amount of dynamin that is recruited to the periactive zone and to scaffold the endocytic process via the interaction with the alpha- and beta-appendages of the clathrin adaptor protein AP2. The AP2-intersectin interaction regulates the ability of intersectin to bind synaptojanin, which promotes uncoating of synaptic vesicles after they have been severed from the plasma membrane. Intersectin and endophilin are required for proper dynamin organization at necks of ccps during clathrin-mediated endocytosis. Endophilin and dynamin assemble into a complex on membrane necks, which is required for fission. Intra-molecular interaction between the SH3- and the F-BAR-domain of syndapin regulates its membrane tubulation activity in vivo and in vitro. This autoinhibition is disrupted by interaction with dynamin. We propose that intersectin, endophilin and syndapin participate in the synaptic vesicle recycling process by regulating the amount of dynamin that is recruited to the periactive zone, by targeting dynamin to necks of ccps and by mediating the assembly of a pre-fission complex, which regulates membrane scission