Fluorescence single-molecule methods represent mighty tools for researchers in the field of structural and molecular biology. These methods are bringing in many advantages when compared to the statistical data processing of multi-molecular species. We can directly compare true statistical distributions and their kinetics. Here belongs fluorescence correlation spectroscopy, FRET and burst variance analysis. As the research advances, new methods are being developed which at the very beginning do not have proper analytical relations for data interpretation and which experimental limits we can't tell. That is the moment when the computer simulations can be used to our advantage. They can help us to specify the right direction of the future research, which is a great money-saver, while opening new perspectives and insights into the explored matter. Correct interpretation of the simulations results is key for the consequent defining of new theoretical models.

The purpose of the study was to investigate why high school junior and senior students in the Jackson Purchase district of Kentucky choose to go or not to go into the profession of teaching Family and Consumer Sciences. The participants of the study were Family and Consumer Sciences teachers and junior and senior high school students enrolled in their classes. This study used a quantitative mode of inquiry. More specifically, two questionnaire instruments were developed and administered to participating teachers and students to answer the research questions. The questionnaire gathered data on the subject's perceptions of Family and Consumer Sciences education. In addition, demographics were derived from student participants. The study determined that students were predominantly college bound, mostly Caucasian, seniors, and female. The majority of students thought Family and Consumer Sciences was important and should be offered in high school. In addition, only 9.3% (10) have considered Family and Consumer Sciences education as a career, 63.7% have never thought of it as a possible career, and 17.8% indicated there was not enough available jobs. The study determined that most of the Family and Consumer Sciences teacher participants declared there was not a decline in enrollment in classes over the past ten years. Two of the seven teachers will be retiring within the next five years.

abstract: Rubisco activase (Rca) from higher plants is a stromal ATPase essential for reactivating Rubiscos rendered catalytically inactive by endogenous inhibitors. Rca’s functional state is thought to consist of ring-like hexameric assemblies, similar to other members of the AAA+ protein superfamily. However, unlike other members, it does not form obligate hexamers and is quite polydisperse in solution, making elucidation of its self-association pathway challenging. This polydispersity also makes interpretation of traditional biochemical approaches difficult, prompting use of a fluorescence-based technique (Fluorescence Correlation Spectroscopy) to investigate the relationship between quaternary structure and function. Like cotton β Rca, tobacco β Rca appears to assemble in a step-wise and nucleotide-dependent manner. Incubation in varying nucleotides appears to alter the equilibrium between varying oligomers, either promoting or minimizing the formation of larger oligomers. High concentrations of ADP seem to favor continuous assembly towards larger oligomers, while assembly in the presence of ATP-yS (an ATP analog) appears to halt continuous assembly in favor of hexameric species. In contrast, assembly in the “Active ATP Turnover” condition (a mixture of ATP and ADP) appears to favor an almost equal distribution of tetramer and hexamer, which when compared with ATPase activity, shows great alignment with maximum activity in the low µM range. Despite this alignment, the decrease in ATPase activity does not follow any particular oligomer, but rather decreases with increasing aggregation, suggesting that assembly dynamics may regulate ATPase activity, rather than the formation/disappearance of one specific oligomer. Work presented here also indicates that all oligomers larger than hexamers are catalytically inactive, thus providing support for the idea that they may serve as a storage mechanism to minimize wasteful hydrolysis. These findings are also supported by assembly work carried out on an Assembly Mutant (R294V), known for favoring formation of closed-ring hexamers. Similar assembly studies were carried out on spinach Rca, however, due to its aggregation propensity, FCS results were more difficult to interpret. Based on these findings, one could argue that assembly dynamics are essential for Rca function, both in ATPase and in regulation of Rubisco carboxylation activity, thus providing a rational for Rca’s high degree of polydispersity. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2018

abstract: Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps. / Dissertation/Thesis / Ph.D. Chemistry 2013

abstract: Ribulose-1, 5-bisphosphate carboxylase oxygenase, commonly known as RuBisCO, is an enzyme involved in carbon fixation in photosynthetic organisms. The enzyme is subject to a mechanism-based deactivation during its catalytic cycle. RuBisCO activase (Rca), an ancillary enzyme belonging to the AAA+ family of the ATP-ases, rescues RuBisCO by facilitating the removal of the tightly bound sugar phosphates from the active sites of RuBisCO. In this work, we investigated the ATP/ADP dependent oligomerization equilibrium of fluorescently tagged Rca for a wide range of concentrations using fluorescence correlation spectroscopy. Results show that in the presence of ADP-Mg2+, the oligomerization state of Rca gradually changes in steps of two subunits. The most probable association model supports the dissociation constants (K_d) of ∼4, 1, 1 μM for the monomer-dimer, dimer-tetramer, and tetramer-hexamer equlibria, respectively. Rca continues to assemble at higher concentrations which are indicative of the formation of aggregates. In the presence of ATP-Mg2+, a similar stepwise assembly is observed. However, at higher concentrations (30-75 µM), the average oligomeric size remains relatively unchanged around six subunits per oligomer. This is in sharp contrast with observations in ADP-Mg2+, where a marked decrease in the diffusion coefficient of Rca was observed, consistent with the formation of aggregates. The estimated K_d values obtained from the analysis of the FCS decays were similar for the first steps of the assembly process in both ADP-Mg2+ and ATP-Mg2+. However, the formation of the hexamer from the tetramer is much more favored in ATP-Mg2+, as evidenced from 20 fold lower K_d associated with this assembly step. This suggests that the formation of a hexameric ring in the presence of ATP-Mg2+. In addition to that, Rca aggregation is largely suppressed in the presence of ATP-Mg2+, as evidenced from the 1000 fold larger K_d value for the hexamer-24 mer association step. In essence, a fluorescence-based method was developed to monitor in vitro protein oligomerization and was successfully applied with Rca. The results provide a strong hint at the active oligomeric structure of Rca, and this information will hopefully help the ongoing research on the mechanistic enzymology of Rca. / Dissertation/Thesis / Ph.D. Chemistry 2014

As the number of people affected by natural and man-made disasters increases, so does the need for emergency preparedness education. Previous research has indicated that education and training can have an impact on the resiliency of individuals, families, and communities. The use of Extension professionals in regional and county offices across each state is an effective means for education. In this study, family and consumer sciences (FCS) county Extension faculty in the Western Region of the United States were surveyed to determine the best practices used and perceptions of the importance and their ability to educate individuals, families, and communities on emergency preparedness. Many respondents agreed on effective strategies to recruit individuals to Extension programming. Overall, FCS county Extension faculty indicated that emergency preparedness being offered was important; however, they did not think they had adequate knowledge or ability to educate on many emergency preparedness topics.

<p>This report primarily focuses on effectively obtaining a Stimulated Emission Depletion fluorescence (STED) microscope, while using Fluorescence Correlation Spectroscopy (FCS) as a guide for the alignment of the system. STED is a super-resolution microscopy technique that has gained favour in the biological sciences due to its ability to successfully resolve sub-diffraction structures within live cells. Moreover the ease with which it can be combined with FCS has extended the applications of this technique to the study of the dynamics within a system as well. The central premise of this work focuses around building a STED-FCS system and developing an alignment tool for obtaining a symmetric STED doughnut. Since the point spread functions (PSF) seen in confocal microscopy can be generally approximated by a Gaussian function, we approximate the doughnut PSF with a difference of Gaussian functions. We calculated an autocorrelation function (ACF) corresponding to the simplified Gaussian form of the doughnut PSF and we found that this ACF contained three very similar diffusion times, all inversely proportional to the dye diffusion coefficient. In agreement with the fact that the doughnut PSF is spread out compared to the purely Gaussian PSF, the doughnut ACF amplitude is lower and its average diffusion time large. Lastly we calculated the quality factor, which is the product of the amplitude of the correlation function with the average intensity, Q=G(0)*I, for the purposes of alignment of the system. When translating the confocal pinhole along an axis of the doughnut we were able to identify the centre of the doughnut due to the presence of a minimum in Q which can be very handy for alignment of the doughnut with respect to the pinhole. This operation is essential when aligning the excitation and STED beam. For future work, a road map for alignment of the two beams in the focal plane is also presented utilizing the cross correlation function between the two beams.</p> / Master of Science (MSc)

No description available.

Fluorescence correlation spectroscopy (FCS) is an ultra-sensitive optical technique to investigate the dynamic properties of ensembles of single fluorescent molecules in solution. It is in particular suited for measurements in biological samples. High sensitivity is obtained by employing confocal microscopy setups with diffraction limited small detection volumes, and by using single-photon sensitive detectors, for example avalanche photo diodes (APD). However, fluorescence signal is hence typically collected from a single focus position in the sample only, and several measurements at different positions have to be performed successively. To overcome the time-consuming successive FCS measurements, we introduce electron multiplying CCD (EMCCD) camera-based spatially resolved detection for FCS. With this new detection method, multiplexed FCS measurements become feasible. Towards this goal, we perform FCS measurements with two focal volumes. As an application, we demonstrate spatial cross-correlation measurements between the two detection volumes, which allow to measure calibration-free diffusion coefficients and direction-sensitive processes like molecular flow in microfluidic channels. FCS is furthermore applied to living zebrafish embryos, to investigate the concentration gradient of the morphogen fibroblast growth factor 8 (Fgf8). It is shown by one-focus APD-based and two-focus EMCCD-based FCS, that Fgf8 propagates largely by random diffusion through the extracellular space in developing tissue. The stable concentration gradient is shown to arise from the equilibrium between a local morphogen production and the sink function of the receiving cells by receptor-mediated removal from the extracellular space. The study shows the applicability of FCS to whole model organisms. Especially in such dynamically changing systems in vivo, the perspective of fast parallel FCS measurements is of great importance. In this work, we exemplify parallel, spatially resolved FCS by utilizing an EMCCD camera. The approach, however, can be easily adapted to any other class of two-dimensional array detector. Novel generations of array detectors might become available in the near future, so that multiplexed spatial FCS could then emerge as a standard extension to classical one-focus FCS. / Fluoreszenz-Korrelations-Spektroskopie (FCS) ist eine hochempfindliche optische Methode, um die dynamischen Eigenschaften eines Ensembles von einzelnen, fluoreszierenden Molekülen in Lösung zu erforschen. Sie ist insbesondere geeignet für Messungen in biologischen Proben. Die hohe Empfindlichkeit wird erreicht durch Verwendung konfokaler Mikroskop-Aufbauten mit beugungsbegrenztem Detektionsvolumen, und durch Messung der Fluoreszenz mit Einzelphotonen-empfindlichen Detektoren, zum Beispiel Avalanche-Photodioden (APD). Dadurch wird das Fluoreszenzsignal allerdings nur von einer einzelnen Fokusposition in der Probe eingesammelt, und mehrfache Messungen an verschiedenen Positionen in der Probe müssen nacheinander durchgeführt werden. Um die zeitaufwendigen, aufeinanderfolgenden FCS-Einzelmessungen zu überwinden, entwickeln wir in dieser Arbeit Elektronenvervielfachungs-CCD (EMCCD) Kamera-basierte räumlich aufgelöste Detektion für FCS. Mit dieser neuartigen Detektionsmethode werden Multiplex-FCS Messungen möglich. Darauf abzielend führen wir FCS Messungen mit zwei Detektionsvolumina durch. Als Anwendung nutzen wir die räumliche Kreuzkorrelation zwischen dem Signal beider Fokalvolumina. Sie ermöglicht die kalibrationsfreie Bestimmung von Diffusionskoeffizienten und die Messung von gerichteter Bewegung, wie zum Beispiel laminarem Fluss in mikrostrukturierten Kanälen. FCS wird darüber hinaus angewendet auf Messungen in lebenden Zebrafischembryonen, um den Konzentrationsgradienten des Morphogens Fibroblasten-Wachstumsfaktor 8 (Fgf8) zu untersuchen. Mit Hilfe von APD-basierter ein-Fokus FCS und EMCCD-basierter zwei-Fokus FCS zeigen wir, dass Fgf8 hauptsächlich frei diffffundiert im extrazellulären Raum des sich entwickelnden Embryos. Der stabile Konzentrationsgradient entsteht durch ein Gleichgewicht von lokaler Morphogenproduktion und globalem Morphogenabbau durch Rezeptor vermittelte Entfernung aus dem extrazellulären Raum. Die Studie zeigt die Anwendbarkeit von FCS in ganzen Modell-Organismen. Gerade in diesen sich dynamisch ändernden Systemen in vivo ist die Perspektive schneller, paralleler FCS-Messungen von großer Bedeutung. In dieser Arbeit wird räumlich aufgelöste FCS am Beispiel einer EMCCD Kamera durchgeführt. Die Herangehensweise ist jedoch einfach übertragbar auf jede andere Art von zwei-dimensionalem Flächendetektor. Neuartige Flächendetektoren könnten in naher Zukunft verfügbar sein. Dann könnte räumlich aufgelöste Multiplex-FCS eine standardisierte Erweiterung zur klassischen ein-Fokus FCS werden.

The cellular membrane is a complex biological entity, far from being an inert assembly of protein and lipids which separates cells from the surrounding environment. A multitude of biological processes, ranging from active transport of ions into and out of the cell, to the immune response, are regulated at the level of the plasma membrane. The understanding of their molecular basis is among the central goals of modern biological research. In order to dissect the complexity of actual cell membranes, which involves a very complex network of intermolecular interactions, a “divide and conquer” strategy proves very useful. To this end, researchers try to isolate molecules from complex biological contexts to understand their function in simple model systems under controlled conditions. A variety of model membranes have thus been developed in order to gain insight into membrane processes. This approach has resulted in a deeper knowledge on how lipids and proteins interact and how these interactions govern the function of cellular membranes. In the recent past in fact, a connection has been established between the lateral structure of the plasma membrane and its biological function. Furthermore, a large range of biophysical techniques have been used to characterize protein-lipid microdomains. For example, atomic force microscopy (AFM) is a powerful technique which allows a highly detailed topographical characterization of lipid domains in physiological conditions. While AFM imaging offers an extremely high spatial resolution, up to the nanometer scale, the limited image acquisition speed (minutes) can pose a severe drawback in adequately studying fast dynamic processes. On the other hand, fluorescence based imaging techniques are much faster (10-3-100 s), but certainly lack the high spatial resolution that AFM offers. FCS in particular can also provide information about dynamic processes, like diffusion of fluorescent membrane components. For these reasons, implementing a combination of the above mentioned techniques on the same sample (e.g. cell membrane models) would prove extremely beneficial in the complete dynamic and structural characterization of molecular interactions. . The work described in this thesis can be summarized in two main points: i) the development of a novel combined approach of atomic force microscopy (AFM), laser scanning imaging (LSM), and fluorescence correlation spectroscopy (FCS) and ii) the study of the effects of ceramide in the lateral organization of model plasma membranes. We described one of the first simultaneous applications of AFM and FCS on biologically relevant systems. More specifically, model membranes showing complex phase separation were investigated with a combined approach of AFM, confocal fluorescence imaging, force measurements and FCS, based on commercially available instruments. AFM conveys information about the structural and mechanical properties of the different lipid phases. Different membrane domains can be distinguished based on height difference, elastic properties and line tension as measured by the AFM tip. Simultaneous optical measurements offer the correlation of these data in real time with the partition behavior and diffusion of fluorescent lipids and proteins. We established a clear link between the local membrane viscosity, probed by FCS, and the lipid-lipid interactions involved in line tension, probed by AFM force measurements. An example of a significant drawback circumvented by the AFM-FCS approach involves the use of AFM micromanipulation to eliminate unwanted interactions between lipid particles — similar to intra-cellular vesicles found in vivo experiments — and the membrane, which usually result in distorted FCS autocorrelation curves. Finally, the combined application of AFM and FCS on membrane-anchored proteins reconstituted in lipid bilayers has been instrumental in clarifying inconsistencies that arose in work that focused solely on either AFM or fluorescence techniques. We have shown that, in the case of proteins diffusing in the plane of the membrane, AFM can unambiguously detect only a small immobile fraction. Furthermore, since AFM detection of proteins might be facilitated by high local membrane viscosity (e.g. in ordered lipid phases), the measurement of protein partition between disordered and ordered membrane domains might be biased toward the latter. In this case, the use of FCS as a complementary technique allows a more thorough investigation and deeper understanding of the system of interest. The second part of this thesis dealt with the study of complex lipid mixtures which are used to model the putative lipid/proteins domains in cells, called “rafts”. Firstly, we proved how the combined fluorescence imaging/AFM approach is useful in general for studying supported lipid membranes and the role of lipid domains in biological contexts. We investigated the effect of environmental stress on biological membranes and the protective effects of several substances. Our experimental approach was shown to be a new valuable method to visualize the dehydration damage and its effects on the lateral organization of lipid domains. Our results demonstrated that disaccharides like trehalose or sucrose are effective in protecting lipid membranes, not only on a macroscopic scale — preserving the overall integrity of the bilayer — but also on a microscopic scale, preventing the clustering of microdomains. These phenomena are interesting in the context of biological damage to living cells which need to be stored for long time, like organs to be transplanted or blood platelets. Finally, a large section of this thesis focused on the effects of a specific lipid called “ceramide” on the lateral organization of proteins and lipids in the plasma membrane. Ceramide is produced by cells in several situations, like bacterial infections or apoptosis. As consequence of ceramide production in vivo, the local concentration and the dynamic behavior of lipids and membrane receptors are supposed to exhibit strong variations. In order to understand the molecular mechanisms responsible for these effects, we applied a combination of AFM, FCS and fluorescence imaging on simple model membranes containing ceramide. We could show for the first time that, in presence of raft-like Lo/Ld phase separation, physiological quantities of ceramide induced the formation of a highly ordered gel phase, constituted of ceramide and sphingomyelin. The enzymatic production of ceramide was monitored both in supported and in free-standing bilayers. In the second case, ceramide production was connected to selective vesicle budding from the raft-like phase. Since short-chain analogues are often used in both medical applications and biochemical research to mimic the effect of long-chain ceramides, we investigated the effect of chain-length on ceramide-induced membrane reorganization. We could show that only long-chain ceramides (C18 and C16) form highly ordered domains. Interestingly, FCS measurements indicated that the physical properties of the Lo raft-like domains are hardly affected by the presence of ceramide domains. Furthermore, the increased thickness of the Ld phase — as measured by AFM — and its higher viscosity — as measured by FCS — strongly support the hypothesis of ceramide-induced cholesterol displacement from rafts. On the other hand, short-chain ceramides showed completely different biophysical properties that lead to a destabilization of the raft domains, possibly acting as surfactants between the different lipid phases. Our findings contribute to the explanation of in vivo experiments where short-chain ceramides inhibit cell signaling by disrupting the lipid order in the plasma membrane. We have so far demonstrated that ceramide plays a fundamental role in lipid-lipid interactions. In a physiological context, it is also known to produce dramatic effects in living cells. Since a majority of the processes in vivo are thought to be governed by the activity of proteins, it is highly likely that ceramide not only affects lipid organization but also modifies protein-protein and protein-lipid interactions to produce its effects. To test this hypothesis, we reconstituted several membrane proteins in lipid bilayers containing Ld, Lo, and ceramide-rich domains. We were able to show that some membrane proteins are sorted into ceramide-rich domains. More specifically, the raft-associated proteins we tested were enriched in the highly ordered ceramide-rich domains, while the Ld-associated components were excluded from them. Furthermore, the inclusion of any membrane component in ceramide-rich domains is directly connected to a dramatic reduction of its in-plane diffusion. In an in vivo context, such a reorganization of membrane receptors might be used by the cell to alter the signaling process, for example, by i) separating raft receptors from inhibitors with lower raft affinity, ii) bringing both raft-associated receptors and raft-associated signaling molecules into contact, or iii) stabilizing the interactions between a receptor and its ligand by decreasing their diffusion coefficients. In conclusion, this thesis describes a novel combination of AFM, LSM, and FCS for the investigation of the lateral organization of biological membranes. Our results show that this approach applied on model membranes of increasing complexity is an effective tool for understanding the molecular mechanisms behind the organization of biological membranes. This report opens up new possibilities for further investigation in living cell membranes using the same methodology we have described.