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Studying Molecular Interactions under Flow with Fluorescence Fluctuation Spectroscopy

dc.contributor.advisorKoester, Sarah Prof. Dr.
dc.contributor.authorPerego, Eleonora
dc.titleStudying Molecular Interactions under Flow with Fluorescence Fluctuation Spectroscopyde
dc.contributor.refereeKoester, Sarah Prof. Dr.
dc.description.abstractengAssembly and interactions of biomolecules are fundamental processes in living organisms to maintain the physiological cellular behavior. The majority of the time protein assembly is useful and vital for the organism. However, sometimes atypical aggregates are produced. These protein aggregates are usually generated from a fault during the normal assembly pathway. Typically, the human body has mechanisms to detect these modifications and discard them, but sometimes the aggregates can be unseen and subsequently lead to pathological situations. A typical example is the aggregation of proteins from the synuclein family, which are considered one of the main factors in the outbreak of some neurodegenerative diseases. It is fundamental to study both the ordered protein assembly and the disordered protein aggregation with spatial resolution on the order of some nm to gain a complete knowledge of the resulting assemblies at a single molecule level. Furthermore, good time resolution of the reaction kinetics, on the order of ms, is also very important. In this thesis, fluorescence fluctuation spectroscopy (FFS) is combined with microfluidics to study protein interactions and assembly with high temporal resolution. FFS is a family of techniques which are all based on studying the fluctuations of the fluorescence signal detected within a confined excitation volume. The fluctuations can be, for example, correlated in time as in fluorescence correlation spectroscopy (FCS) to obtain information about protein mobility. Alternatively, the frequency of the fluctuations can be evaluated, as in photon counting histogram (PCH) to measure the brightness of the fluorescence molecules. FFS is often applied to quantify aggregation, however, all the techniques from this family lack high temporal resolution, since long acquisition times are usually needed for a good signal-to-noise ratio. Thus, microfluidic methods are integrated into our measurements to access different time points in protein reactions. Here, continuous flow microfluidics is employed to track the early time points of vimentin assembly. A five-inlet step microfluidic device compatible with microscopy and FFS is manufactured. The adsorption of assembling vimentin on the channel walls is avoided by creating a step in the central channel, which helps to encapsulate the vimentin jet into a buffer protection layer and prevents the protein from touching the device walls. The protein assembly is initiated by diffusion mixing vimentin tetramers and assembly buffer. Vimentin lateral assembly is quantified and compared with the current state of the art. Our measurements of vimentin lateral assembly can be used to fill the gap of the early time points in the overall dynamics of vimentin. Synaptic protein interactions are also quantified by combining microfluidics and FFS. A bottom up approach is presented here to study synaptic interaction in a controlled environment, in particular a simpler version of the synaptic environment is reconstituted on a substrate by patterning SVs on glass coverslips. We show that the interactions between synaptic proteins and SV can be quantified with FCS. A microfluidic chamber is furthermore built, compatible with patterned molecules, to allow us to access also the temporal information during synaptic interactions. We showed that the combination of microfluidics and FFS techniques can be applied in general to study the dynamic of various processes in biology, varying from cytoskeleton assembly to synaptic protein
dc.contributor.coRefereeRizzoli, Silvio Prof. Dr.
dc.contributor.thirdRefereeGriesinger, Christian Prof. Dr.
dc.contributor.thirdRefereeKruss, Sebastian Dr.
dc.contributor.thirdRefereeAdio, Sarah Dr.
dc.contributor.thirdRefereeGrubmüller, Helmut Prof. Dr.
dc.subject.engFluorescence spectroscopyde
dc.subject.engDynamics of protein assemblyde
dc.subject.engSynaptic protein dynamicsde
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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