The intensity of light produced from synchrotrons enables X-ray imaging down to the micron and submicron scale. This high degree of resolution is necessary to study metals in hydrated biological samples, where trace (metal) elements are found in the lowest concentration. Water within these aqueous samples will undergo radiolysis and produce various reactive oxygen species, which degrades the quality of information gathered from the sample during X-ray imaging. Studies have shown that the best way to counter the effects of radiolysis and preserve samples in their metabolic state during X-ray imaging is to keep them cryogenically frozen. We have developed affordable cryostats and novel protocol to not only improve cryo-imaging at current third-generation synchrotrons, but also enable cryo-imaging at existing synchrotrons that have limited accessibility. This dissertation will provide a detailed description of the tasks that were accomplished to contribute to the development cryo-imaging. The first task was the fabrication of a portable cryostage. The cryostage's discreet profile and unique design successfully enabled it to be effortlessly adapted into three beamlines across two different DOE facilities and facilitate multiple imaging modalities, i.e., correlative imaging. With the next task, we explored adding an ice frame about the stage to help reduce the accumulation of frost on the surface of a frozen sample that was explored. The addition of the ice frame significantly improved the imaging of frozen samples, nearly doubling the overall image clarity in comparison to when it was absent. The final task saw the application of a cryostream, in place of a cryostage, to provide a cooled convective flux across the sample for 2D and 3D visualization for cryo X-ray imaging. / Doctor of Philosophy / Synchrotrons are light producing particle accelerators that enable X-ray imaging down to the micron and submicron scale. This high degree of resolution is necessary to study metals in hydrated biological samples, where trace elements are found in low concentrations. The X-ray beam from the synchrotron will force any water within these aqueous samples to undergo radiation induced water decomposition, i.e., radiolysis, and produce hydroxyl radicals that will degrade the quality of information gathered from the sample during X-ray imaging. Early studies have shown that the best counter to the effects of radiolysis, while also preserving samples in their metabolic state during X-ray imaging, is to keep them cryogenically frozen. We have developed affordable cryostats and novel protocols to not only improve cryo-imaging at current third-generation synchrotrons, but also enable cryo-imaging at existing synchrotrons that have limited accessibility. This dissertation will describe, in detail, three tasks that were accomplished. The first task was to the fabrication of a portable cryostage. The cryostage unique design successfully enabled it to be used within different beamlines and for multiple imaging perspectives. With the next task, an ice frame to help reduce the accumulation of frost on the surface of a frozen sample being explored. The ice frame was shown to significantly improve the imaging of frozen samples. The final task saw the application of a cryostream, a jet stream of cold nitrogen gas, to enable an alternative approach for 2D and 3D visualization for cryo X-ray imaging.

Due to the limited supply and perishable nature of blood products, effective management of blood collection is critical for high quality healthcare delivery. Whole blood is typically collected over a 6 to 8 hour collection window from volunteer donors at sites, e.g., schools, universities, churches, companies, that are a significant distance from the blood products processing facility and then transported from collection site to processing facility by a blood mobile. The length of time between collecting whole blood and processing it into cryoprecipitate ("cryo"), a critical blood product for controlling massive hemorrhaging, cannot take longer than 8 hours (the 8 hour collection to completion constraint), while the collection to completion constraint for other blood products is 24 hours. In order to meet the collection to completion constraint for cryo, it is often necessary to have a "mid-drive collection"; i.e., for a vehicle other than the blood mobile to pickup and transport, at extra cost, whole blood units collected during early in the collection window to the processing facility. In this dissertation, we develop analytical models to: (1) analyze which collection sites should be designated as cryo collection sites to minimize total collection costs while satisfying the collection to completion constraint and meeting the weekly production target (the non-split case), (2) analyze the impact of changing the current process to allow collection windows to be split into two intervals and then determining which intervals should be designated as cryo collection intervals (the split case), (3) insure that the weekly production target is met with high probability. These problems lead to MDP models with large state and action spaces and constraints to guarantee that the weekly production target is met with high probability. These models are computationally intractable for problems having state and action spaces of realistic cardinality. We consider two approaches to guarantee that the weekly production target is met with high probability: (1) a penalty function approach and (2) a chance constraint approach. For the MDP with penalty function approach, we first relax a constraint that significantly reduces the cardinality of the state space and provides a lower bound on the optimal expected weekly cost of collecting whole blood for cryo while satisfying the collection to completion constraint. We then present an action elimination procedure that coupled with the constraint relaxation leads to a computationally tractable lower bound. We then develop several heuristics that generate sub-optimal policies and provide an analytical description of the difference between the upper and lower bounds in order to determine the quality of the heuristics. For the multiple decision epoch MDP model with chance constraint approach, we first note by example that a straightforward application of dynamic programming can lead to a sub-optimal policy. We then restrict the model to a single decision epoch. We then use a computationally tractable rolling horizon procedure for policy determination. We also present a simple greedy heuristic (another rolling horizon decision making procedure) based on ranking the collection intervals by mid-drive pickup cost per unit of expected cryo collected, which results in a competitive sub-optimal solution and leads to the development of a practical decision support tool (DST). Using real data from the American Red Cross (ARC), we estimate that this DST reduces total cost by about 30% for the non-split case and 70% for the split case, compared to the current practice. Initial implementation of the DST at the ARC Southern regional manufacturing and service center supports our estimates and indicates the potential for significant improvement in current practice.

Cette thèse porte sur une étude expérimentale et théorique de surfaces micro-structurées de silicium, obtenues par traitement dans un plasma SF6/02 à des températures cryogéniques. La texturation qui résulte de ce traitement confère à ces surfaces des propriétés remarquables. L'une d'entre elles est la capacité de piéger et absorber la lumière, qui se traduit par une couleur noire de ces surfaces, d'où l'appellation Black Silicon. Cette propriété qu'on retrouve dans la gamme spectrale du visible et du proche infra-rouge, présente un intérêt particulier pour la conversion d'énergie solaire, aussi bien par voie photovoltaïque que par voie photo-thermique. L'étude que nous avons menée a toutefois porté sur une gamme spectrale plus large, s'étendant jusqu'aux Térahertz. A cet effet, différentes techniques de caractérisation spectrales ont été mises en œuvre. L'analyse des résultats a été effectuée également au moyen de simulations électromagnétiques. Des corrélations ont été mises en évidence entre les propriétés optiques et les caractéristiques morphologiques des surfaces micro-structurées. L'analyse d'images prises au microscope électronique a permis d'esquisser une théorie pour tenter d'expliquer le mécanisme de formation des microstructures de Black Silicon. Enfin, un microcomposant a été réalisé en vue de mettre en œuvre le premier volet applicatif de ce travail. Il s'agit d'un dispositif de conversion photo-thermique qui incorpore des thermo-résistances en platine sur une surface de Black Silicon réalisée sur une membrane thermiquement isolée / This thesis deals with an experimental and theoretical study of micro-structured silicon surfaces, obtained by processing in SF6/02 plasma at cryogenic temperatures. Texturing which results from this treatment gives remarkable properties to these surfaces. One of them is the ability to trap and absorb light, resulting in a black color of the surface, hence the name of Black Silicon. This property that we find in the visible and near infrared spectral ranges, is of particular interest for solar energy conversion, both through photovoltaic and photo-thermal means. The study that we conducted, however, covered a much wider spectral range, extending to the Terahertz. For this purpose, different spectral characterization techniques have been implemented. Analysis of the results was also done using electromagnetic simulations. Correlations were found between the optical and morphological characteristics of micro-structured surfaces. The analysis of images taken by electron microscopy allowed sketching a theory attempting to explain the mechanism of formation of the microstructures of Black Silicon. Finally, a micro-component has been fabricated towards implementing the first part of this application work. It is a photo-thermal conversion device that incorporates platinum thermo-resistance on a surface of Black Silicon, realized on a thermally insulated Silicon membrane

Helical assemblies of proteins are ubiquitous in nature and they perform vital functions in a wide range of organisms. The recent development of direct electron detectors and other imaging techniques in cryo-electron microscopy (cryo-EM) has opened new possibilities in solving helical structures at atomic resolution. Existing software packages for helical processing often require experience in tuning many ad hoc parameters to achieve optimal reconstruction results. REgularised LIkelihood OptimisatioN (RELION), an open-source single-particle analysis package, reduces the need for user expertise by the formulation of an empirical Bayesian framework, and has yielded some of the highest resolution density maps in recent years. Prior information about the helical assemblies can be conveniently incorporated into the statistical framework of RELION and thereby improves the helical reconstructions. This PhD thesis describes the development of a helical processing computation workflow with reduced user intervention in RELION. Chapter 1 introduces the theoretical basis of cryo-EM data acquisition and single-particle data processing, the concepts of helical symmetry, and a previously described method for iterative real-space reconstruction of helical assemblies, to which the RELION implementation bears resemblance. Chapter 2 discusses multiple adaptations to RELION that are necessary for helical processing. Key elements include the imposition and local refinement of helical symmetry, masks on helical segments and references, expressions of angular and translational prior information, manual and automated segment picking as well as initial model generation for helices. Calculations have been performed on four test data sets showing that the developed methods in RELION yield results that are as good as or better than alternative approaches for the tests performed. Chapter 3 describes the same methodology adapted to helical sub-tomogram averaging in RELION. Chapter 4 introduces the local symmetry option developed for special types of filaments with pseudo-helical symmetry. The concept can be extended to general single-particle analysis as well. Chapter 5 describes four helical structures determined in collaboration with other research groups using helical RELION for data processing. Chapter 6 concludes the thesis with a brief summary and future prospects.

The main objective of this work is to determine the atomic structure of mammalian respiratory complex I. Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) is one of the central enzymes in the oxidative phosphorylation pathway. It couples electron transfer between NADH and ubiquinone to proton translocation across the inner mitochondrial membrane, contributing to cellular energy production. Complex I is the largest and most elaborate protein assembly of the respiratory chain with a total mass of 970 kilodaltons. It consists of 14 conserved ‘core subunits’ and 31 mitochondria-specific ‘supernumerary subunits’. Together they form a giant, Lshaped molecule, with one arm buried in the mitochondrial membrane and another protruding into the mitochondrial matrix. Here, a novel method for the purification of ovine (Ovis aries) complex I was developed and suitable conditions for cryo-EM imaging established, after extensive screening of detergents and additives. Cryo-EM images were acquired with the recently developed direct electron detector and processed using the latest software. This allowed the solution of the nearly complete atomic model of mitochondrial complex I at 3.9 Å resolution. The membrane part of the complex contains 78 transmembrane helices, mostly contributed by conserved antiporter-like subunits responsible for proton translocation. These helices are stabilized by tightly bound lipids (including cardiolipins). The hydrophilic arm harbours flavin mononucleotide and 8 iron–sulfur clusters involved in electron transfer. Supernumerary subunits build a scaffold around the conserved core, strongly stabilizing the complex. Additionally, subunits containing cofactors (NADPH, zinc ion and phosphopantetheine) may play a regulatory role. Two distinct conformations of the complex are observed, which may describe the active and deactive states or reflect conformations occurring during the catalytic cycle of the enzyme. Currently this is the most detailed model of this molecular machine, providing insight into the mechanism, assembly and dysfunction of mitochondrial complex I. It also allows molecular analysis of numerous disease-causing mutations, and so the structure may serve as a stepping-stone for future medical developments.

Cdc48A and its human homologue P97 are from ATPase family, which play a variety of roles in cellular activates and it has a crucial involvement in protein quality control pathways. It is best known for its involvement in endoplasmic reticulum associated protein degradation (ERAD), where it mediates the degradation of the aggerated or misfolded proteins by the proteasome. Considering the multiple functions of Cdc48A in many protein regulatory processes, it is a potential therapeutic target for neurogenerative diseases and cancer. Cdc48A polypeptide comprises N domain, followed by D1 and D2 domains respectively that are joined by linkers, whereas functionally it forms a homo hexameric complex. Since Cdc48A is from the ATPase family, it uses the ATP hydrolysis to generate a mechanical force with its co-factors to perform its functions. There are many cofactors that interact with Cdc48A and two of them are Ufd1-NpI4 which in turn interact with ubiquitinated proteins from the ER membrane. The mechanism linking the conversion of the energy of ATP hydrolysis into mechanical force and unfolding the substrate is vague. My aim is to reconstruct a first 3D- model of plant Cdc48A using single particle cryo-EM, which serves the basis to conduct more detailed mechanistic studies towards substrate unfolding and threading/unfolding in the future. In general, results showed one defined structure of cdc48A at ~ 9.8 Å, which is the ADP-ATP conformation. Although another other structure was also resolved at ~ 8.9 Å, it was hard to characterize due to its dissimilarity with known structures of Cdc48A homologues and thus requires further characterization.

Legionella pneumophila (Lp) is a gram-negative, intracellular parasite. These bacteria evade the host response with the help of a Type IVb secretion system (T4bSS), composed of Defective in organelle trafficking (Dot) and Intracellular multiplication (Icm) proteins. This secretion system delivers over 300 effectors into the host, and a large number of these molecules are dependent on IcmS and IcmW. These effectors are essential for the bacterium’s survival in the host. This work and previous studies have shown that IcmS and IcmW interact to stabilize each other and the C-terminal "tail" of DotL (residues 655-783), a coupling protein in the T4bSS, binds to the IcmSW complex to further increase its stability. All three components are α-helical, making the complex amenable to structural studies by X-ray crystallography and cryo-electron microscopy. Three maps of the IcmSW-DotL-tail complex (~42 kDa) were generated from cryo-EM images recorded with a Volta phase plate and K2 Summit direct electron detector at 500-1000 nm under-defocus. The final maps were processed with RELION-2 and resolved to 5.5-6.5 Å resolution using 57k, 60k and 80k particles, respectively. Concurrent with work in this thesis, a crystal structure of IcmSW-DotL(656-783) was solved by Dr. Byung-Ha Oh’s group at KAIST. This structure was used as a comparative model for our cryo-EM 3D reconstructions which were determined to evaluate size limits imposed on single particle methods with current technology and to provide snapshots of the complex in solution. Comparisons between the crystal structure and cryo-EM maps show that the overall structure is similar in solution, but there is significant flexibility within each subunit with a repositioning of some α-helices and surface loops. Flexibility in the absence of a central subunit (LvgA), and a low number of good particles may have limited the final resolution. Although the current maps were determined at α-helical resolution, this work provides a road map for solving near atomic structures at or near the size of IcmSW-DotL-tail. This structural technology will provide a means to probe the solution structure and function of biological machines in a large range of sizes and conformations.

Bovine embryos produced in vitro consistently display decreased quality in terms of their potential to reach the blastocyst stage as well as post-transfer survival. Media formulations, oocyte quality, and inferior expression of needed transcripts are all causes of this reduced developmental potential commonly present in in vitro-produced (IVP) bovine embryos. Recently our lab has confirmed interleukin-6 as an embryokine whose capabilities include increasing inner cell mass (ICM) numbers and promoting bovine blastocyst development. LIF is another family member of the IL6 cytokine family and has been shown to produce several positive effects when supplemented during oocyte in vitro maturation. IL6 has predominantly been studied as being supplemented post-fertilization. However, published transcriptomic work described receptors for IL6, IL11, and LIF as present in cumulus cells at the time COCs are removed from their follicles. Consequently, we wanted to investigate if supplementing 25 ng/ml of IL6, IL11, or LIF would improve IVM bovine oocyte competency. Several experiments were completed (4replicates/experiment; 30-60 cumulus-oocyte complexes (COCs)/replicate). In Experiment 1, COCs were in vitro matured for 16 or 22 hours, then meiotic stage was assessed after denuding, fixation, and DNA staining. No cytokine treatment influenced the percentage of oocytes that achieved metaphase II at either time point. In Experiment 2, COCs were in vitro matured for 4 hours before snap freezing. and processing to examine changes in five cumulus-expressing transcripts associated with oocyte competency (CX43, CX37, AREG, TNFAIP6, HAS2). Our chosen housekeeping gene, HPRT1, served as the internal control. An increased abundance of AREG occurred following exposure to LIF but not with the other treatments. Supplementation with IL6 and IL11 but not LIF tended to increase TNFAIP6 abundance (P<0.10). No other transcript differences were detected. In Experiment 3, we examined whether supplementing these cytokines during IVM affects subsequent fertilization and blastocyst rates. No effects were detected on cleavage rates but at day 8 increases in blastocyst yield were detected for LIF and IL11, but not IL6. LIF showed a tendency to increase hatching rates. In Experiment 4, we aimed to assess how the cytokine treatments affected cryosurvivability. Blastocysts (5-10/replicate, 9 replicate studies) were frozen at a rate of -0.6 degrees C/min until reaching -32 degrees C, then were stored in liquid nitrogen for 4-8 weeks before being thawed and incubated in conventional embryo culture medium (SOF-BE1) for 3 days. No treatment effects were noted for re-expansion, hatching, and overall survivability. In summary, these results implicate IL11 and LIF as potential mediators of oocyte competency. However, the evidence presented here suggest that IL6 and IL11 may function differently than LIF when provided during COC maturation. / Master of Science / The numerous similarities in the regulation of early embryonic development between human and cow has made bovine embryos an excellent model for exploring how to optimize assisted reproductive techniques (ARTs) and other methods for improving and preserving fertility in humans. Pregnancy loss is also very similar in both cattle and humans. In beef cattle, more than 50 percent of reproductive failures occur before day 16 of gestation. In women, approximately 15 percent of all clinically recognized pregnancies result in spontaneous loss, however, several more pregnancies fail prior to ever being clinically recognized. Various ARTs are used to treat sub-fertile conditions in cattle, and these technologies are generally deemed as a viable way to improve fertility. However, IVP embryos are inferior in their ability to properly fertilize and develop to the blastocyst stage, the stage when embryos are normally transferred. Furthermore, IVP-generated embryos are inferior at maintaining pregnancies. There are two primary restraints to the IVP process: a low percentage of oocytes that become fertilized and produce transferable embryos and transferred IVP embryos have decreased chances of maintaining a viable embryo than embryos produced in vivo. Very little is known about the various hormone and molecular factors that promote oocyte and embryo development. Therefore, a primary objective for bovine oocyte and embryo research is to classify these factors and implement them into their maturation and culture media to improve overall IVP efficiency. My lab studies members of the IL6 cytokine family as potential factors that might play a role in the development of oocytes and embryos. The aim of this work is to assess the capacity of three molecules within this family, IL6, IL11, and Leukemia inhibitory factor (LIF) to improve oocyte development, fertilization rate, and blastocyst yield when supplemented during in vitro maturation (IVM). This work revealed that both IL11 and LIF improved IVP bovine blastocyst development at day 8. Unfortunately, none of the treatments had any effect on fertilization rates. LIF increased the expression of a cumulus-specific transcript known to aid in cumulus expansion. Cumulus cells are the somatic cells immediately surrounding the oocyte. Cumulus expansion is a key indicator of proper oocyte maturation. We did not observe any treatment effect on post-thaw survival of cryopreserved bovine embryos. This indicates that our treatments did not help the embryos maintain viability after undergoing a slow-freeze cryopreservation protocol followed by thawing and culture. In summary, this work showed that IL11 and LIF have potential benefits to the in vitro production of bovine embryos when supplemented at IVM. However, future work is needed to assess how these molecules are causing these improvements. Our results indicate that IL11 and LIF may function differently from IL6 when supplemented during IVM.

Characterization of Rubisco plays a crucial role when it comes to the development and understanding of carbon sequestration in plants. This project took place at BMC in Uppsala, in the Gunn lab, and aimed to structure three Rubisco structures and analyze these with regard to the assembly pathway of the biogenesis of Rubisco but also how fast the reaction of binding of atmospheric carbon dioxide takes place with regard to different isoforms of the small subunit. The structural regulations led to the conclusion that an additional step in the assembly pathway would be added when one side of Rubisco had the chaperone BSD2 bound while the other side of Rubisco had the small subunit bound.The different subunits are believed to effect the structure of the LSu. The result also indicate that when the SSu are binding to the LSu octomer the interactions between the BSD2 and the LSu changes. This indicats that the SSu could indirectly facilitate the binding of the SSu on the other side by affecting the interactions of the LSu and the BSD2. Therefore the cooperative binding of the different subunits would be interesting to further evaluate. The NtL8B4(S-T1)4 , which is the first model for this structure to be determined, and therefore extended the assembly pathway for the biogenesis of higher plants, had the CABP bound, indicating that this intermediate structure could be analytically competent. This hypothesis is only based on the analyses of the structural determination, therefore further studies are needed to determine whether this is legitimate. Teknisk-naturvetenskapliga fakulteten, Uppsala universitet. Utgivni

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