Search

Inhalation exposure to environmental and occupational aerosol contaminants is associated with many respiratory health problems. To realistically mimic long-term inhalation exposure for toxicity testing, lung epithelial cells need to maintained and exposed under air-liquid interface (ALI) conditions for a prolonged period of time. In addition, to study cellular responses to aerosol particles, lung epithelial cells have to be co-cultured with macrophages. To that aim, we evaluated human bronchial epithelial Calu-3, 16HBE14o- (16HBE), H292, and BEAS-2B cell lines with respect to epithelial morphology, barrier function and cell viability under prolonged ALI culture conditions. Only the Calu-3 cells can retain the monolayer structure and maintain a strong tight junction under long-term ALI culture at least up to 2 weeks. As such, Calu-3 cells were applied as the structural barrier to create co-culture models with human monocyte-derived macrophages (MDMs) and THP-1 derived macrophages (TDMs). Adhesion of macrophages onto the epithelial monolayer was allowed for 4 h with a density of 5 × 104 macrophages/cm2. In comparison to the Calu-3 mono-culture model, Calu-3 + TDM and Calu-3 + MDM co-culture models showed an increased sensitivity in inflammatory responses to lipopolysaccharide (LPS) aerosol at Day 1 of co-culture, with the Calu-3 + MDM model giving a stronger response than Calu-3 + TDM. Therefore, the epithelial monolayer integrity and increased sensitivity make the Calu-3 + MDM co-culture model a preferred option for ALI exposure to inhaled aerosols for toxicity testing.

Purpose The aim of this work was to develop a CAIX-specific nanobody conjugated to IRDye800CW for molecular imaging of pre-invasive breast cancer. Procedures CAIX-specific nanobodies were selected using a modified phage display technology, conjugated site-specifically to IRDye800CW and evaluated in a xenograft breast cancer mouse model using ductal carcinoma in situ cells (DCIS). Results Specific anti-CAIX nanobodies were obtained. Administration of a CAIX-specific nanobody into mice with DCIS xenografts overexpressing CAIX showed after 2 h a mean tumor-to-normal tissue ratio (TNR) of 4.3 ± 0.6, compared to a TNR of 1.4 ± 0.2 in mice injected with the negative control nanobody R2-IR. In DCIS mice, a TNR of 1.8 ± 0.1 was obtained. Biodistribution studies demonstrated an uptake of 14.0 ± 1.1 %I.D./g in DCIS + CAIX tumors, 4.6 ± 0.8 %I.D./g in DCIS tumors, while 2.0 ± 0.2 %I.D./g was obtained with R2-IR. Conclusions These results demonstrate the successful generation of a CAIX-specific nanobody-IRDye800CW conjugate that can be used for rapid imaging of (pre-)invasive breast cancer. Electronic supplementary material The online version of this article (doi:10.1007/s11307-015-0909-6) contains supplementary material, which is available to authorized users.

The rapidly expanding manufacturing, production and use of nanomaterials have raised concerns for both worker and consumer safety. Various studies have been published in which induction of pulmonary inflammation after inhalation exposure to nanomaterials has been described. Nanomaterials can vary in aspects such as size, shape, charge, crystallinity, chemical composition, and dissolution rate. Currently, efforts are made to increase the knowledge on the characteristics of nanomaterials that can be used to categorise them into hazard groups according to these characteristics. Grouping helps to gather information on nanomaterials in an efficient way with the aim to aid risk assessment. Here, we discuss different ways of grouping nanomaterials for their risk assessment after inhalation. Since the relation between single intrinsic particle characteristics and the severity of pulmonary inflammation is unknown, grouping of nanomaterials by their intrinsic characteristics alone is not sufficient to predict their risk after inhalation. The biokinetics of nanomaterials should be taken into account as that affects the dose present at a target site over time. The parameters determining the kinetic behaviour are not the same as the hazard-determining parameters. Furthermore, characteristics of nanomaterials change in the life-cycle, resulting in human exposure to different forms and doses of these nanomaterials. As information on the biokinetics and in situ characteristics of nanomaterials is essential but often lacking, efforts should be made to include these in testing strategies. Grouping nanomaterials will probably be of the most value to risk assessors when information on intrinsic characteristics, life-cycle, biokinetics and effects are all combined.

A combination of atomic force microscopy (AFM), high-resolution scanning electron microscopy (HR-SEM), focused-ion-beam scanning electron microscopy (FIB-SEM), X-ray photoelectron spectroscopy (XPS), confocal fluorescence microscopy (CFM), and UV/Vis and synchrotronbased IR microspectroscopy was used to investigate the dealumination processes of zeolite ZSM-5 at the individual crystal level. It was shown that steaming has a significant impact on the porosity, acidity, and reactivity of the zeolite materials. The catalytic performance, tested by the styrene oligomerization and methanol-to-olefin reactions, led to the conclusion that mild steaming conditions resulted in greatly enhanced acidity and reactivity of dealuminated zeolite ZSM-5. Interestingly, only residual surface mesoporosity was generated in the mildly steamed ZSM- 5 zeolite, leading to rapid crystal coloration and coking upon catalytic testing and indicating an enhanced deactivation of the zeolites. In contrast, harsh steaming conditions generated 5–50 nm mesopores, extensively improving the accessibility of the zeolites. However, severe dealumination decreased the strength of the Brønsted acid sites, causing a depletion of the overall acidity, which resulted in a major drop in catalytic activity.

Autotransporters produced by Gram-negative bacteria consist of an N-terminal signal sequence, a C-terminal translocator domain (TD), and a passenger domain in between. The TD facilitates the secretion of the passenger across the outer membrane. It generally consists of a channel-forming β-barrel that can be plugged by an α-helix that is formed by a polypeptide fragment immediately N-terminal to the barrel domain in the sequence. In this work, we characterized the TD of the hemoglobin protease Hbp of Escherichia coli by comparing its properties with the TDs of NalP of Neisseria meningitidis and IgA protease of Neisseria gonorrhoeae. All TDs were produced in inclusion bodies and folded in vitro. In the case of the TD of Hbp, this procedure resulted in autocatalytic intramolecular processing, which mimicked the in vivo processing. Liposome-swelling assays and planar lipid bilayer experiments revealed that the pore of the Hbp TD was largely obstructed. In contrast, an Hbp TD variant that lacked only one amino-acid residue from the N terminus showed the opening and closing of a channel comparable to what was reported for the TD of NalP. Additionally, the naturally processed helix contributed to the stability of the TD, as shown by chemical denaturation monitored by tryptophan fluorescence. Overall these results show that Hbp is processed by an autocatalytic intramolecular mechanism resulting in the stable docking of the α-helix in the barrel. In addition, we could show that the α-helix contributes to the stability of TDs. © 2011 Informa UK, Ltd.

Immuno-transmission electron microscopy (TEM) is the technique of choice for high-resolution localization of proteins in fixed specimen. Here we introduce 2 novel methods for the fixation of sections from cryo-immobilized samples that result in excellent ultrastructural preservation. These high-speed fixation techniques, both called VIS2FIX, allow for a reduction in sample preparation time from at least 1 week to only 8 h. The methods were validated in immuno-TEM experiments on THP-1 monocytes, human umbilical vein endothelial cells (HUVECs) and Madin–Darby canine kidney (MDCK-II) cells. The fixation and retention of neutral lipids is demonstrated, offering unique prospects for the application of immuno-TEM in the lipidomics field. Furthermore, the VIS2FIX methods were successfully employed in correlative fluorescence and electron microscopy.

Break on through: Steaming-induced mesopores of individual ZSM-5 crystals were studied by a combination of focused ion beam (FIB) and scanning electron microscopy (SEM) tomography (see picture). In this manner, quantitative insight into the width, length, morphology, and distribution of mesopores generated within zeolite crystals has been obtained. Keywords:crystal intergrowth;scanning probe microscopy;mesoporosity;tomography;zeolites

Understanding the internal mechanisms controlling fault friction is crucial for understanding seismogenic slip on active faults. Displacement in such fault zones is frequently localized on highly reflective (mirrorlike) slip surfaces, coated with thin films of nanogranular fault rock. We show that mirror-slip surfaces developed in experimentally simulated calcite faults consist of aligned nanogranular chains or fibers that are ductile at room conditions. These microstructures and associated frictional data suggest a fault-slip mechanism resembling classical Ashby-Verrall superplasticity, capable of producing unstable fault slip. Diffusive mass transfer in nanocrystalline calcite gouge is shown to be fast enough for this mechanism to control seismogenesis in limestone terrains. With nanogranular fault surfaces becoming increasingly recognized in crustal faults, the proposed mechanism may be generally relevant to crustal seismogenesis.

BACKGROUND: Mycelia of higher fungi consist of interconnected hyphae that are compartmentalized by septa. These septa contain large pores that allow streaming of cytoplasm and even organelles. The cytoplasm of such mycelia is therefore considered to be continuous. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show by laser dissection that septa of Schizophyllum commune can be closed depending on the environmental conditions. The most apical septum of growing hyphae was open when this basidiomycete was grown in minimal medium with glucose as a carbon source. In contrast, the second and the third septum were closed in more than 50% and 90% of the cases, respectively. Interestingly, only 24 and 37% of these septa were closed when hyphae were growing in the absence of glucose. Whether a septum was open or closed also depended on physical conditions of the environment or the presence of toxic agents. The first septum closed when hyphae were exposed to high temperature, to hypertonic conditions, or to the antibiotic nourseothricin. In the case of high temperature, septa opened again when the mycelium was placed back to the normal growth temperature. CONCLUSIONS/SIGNIFICANCE: Taken together, it is concluded that the septal pores of S. commune are dynamic structures that open or close depending on the environmental conditions. Our findings imply that the cytoplasm in the mycelium of a higher fungus is not continuous per se.

Coronaviruses are enveloped viruses containing the largest reported RNA genomes. As a result of their pleomorphic nature, our structural insight into the coronavirion is still rudimentary, and it is based mainly on 2D electron microscopy. Here we report the 3D virion structure of coronaviruses obtained by cryo-electron tomography. Our study focused primarily on the coronavirus prototype murine hepatitis virus (MHV). MHV particles have a distinctly spherical shape and a relatively homogenous size (approximate to 85 nm envelope diameter). The viral envelope exhibits an unusual thickness (7.8 +/- 0.7 nm), almost twice that of a typical biological membrane. Focal pairs revealed the existence of an extra internal layer, most likely formed by the C-terminal domains of the major envelope protein M. In the interior of the particles, coiled structures and tubular shapes are observed, consistent with a helical nucleocapsid model. Our reconstructions provide no evidence of a shelled core. Instead, the ribonucleoprotein seems to be extensively folded onto itself, assuming a compact structure that tends to closely follow the envelope at a distance of approximate to 4 nm. Focal contact points and thread-like densities connecting the envelope and the ribonucleoprotein are revealed in the tomograms. Transmissible gastroenteritis coronavirion tomograms confirm all the general features and global architecture observed for MHV. We propose a general model for the structure of the coronavirion in which our own and published observations are combined.

Colonies of Aspergillus niger are characterized by zonal heterogeneity in growth, sporulation, gene expression and secretion. For instance, the glucoamylase gene glaA is more highly expressed at the periphery of colonies when compared to the center. As a consequence, its encoded protein GlaA is mainly secreted at the outer part of the colony. Here, multiple copies of amyR were introduced in A. niger. Most transformants over-expressing this regulatory gene of amylolytic genes still displayed heterogeneous glaA expression and GlaA secretion. However, heterogeneity was abolished in transformant UU-A001.13 by expressing glaA and secreting GlaA throughout the mycelium. Sequencing the genome of UU-A001.13 revealed that transformation had been accompanied by deletion of part of the fluG gene and disrupting its 3' end by integration of a transformation vector. Inactivation of fluG in the wild-type background of A. niger also resulted in breakdown of starch under the whole colony. Asexual development of the ∆fluG strain was not affected, unlike what was previously shown in Aspergillus nidulans. Genes encoding proteins with a signal sequence for secretion, including part of the amylolytic genes, were more often downregulated in the central zone of maltose-grown ∆fluG colonies and upregulated in the intermediate part and periphery when compared to the wild-type. Together, these data indicate that FluG of A. niger is a repressor of secretion.

Insight into processes leading to rupture of intracranial aneurysms (IAs) may identify biomarkers for rupture or lead to management strategies reducing the risk of rupture. We characterized and quantified (ultra)structural differences between unruptured and ruptured aneurysmal walls. Six unruptured and 6 ruptured IA fundi were resected after microsurgical clipping and analyzed by correlative light microscopy for quantitative analysis (proportion of the vessel wall area) and transmission electron microscopy for qualitative ultrastructural analysis. Quantitative analysis revealed extensive internal elastic lamina (IEL) thickening in ruptured IA (36.3% ± 15%), while thin and fragmented IEL were common in unruptured IA (5.6% ± 7.1%). Macrophages were increased in ruptured IA (28.3 ± 24%) versus unruptured IA (2.7% ± 5.5%), as were leukocytes (12.85% ± 10% vs 0%). Vasa vasorum in ruptured but not in unruptured IA contained vast numbers of inflammatory cells and extravasation of these cells into the vessel wall. In conclusion, detection of thickened IEL, leaky vasa vasorum, and heavy inflammation as seen in ruptured IA in comparison to unruptured IA may identify aneurysms at risk of rupture, and management strategies preventing development of vasa vasorum or inflammation may reduce the risk of aneurysmal rupture.

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM.

In recent years, electron tomography has improved our three-dimensional (3D) insight in the structural architecture of cells and organelles. For studies that involve the 3D imaging of stained sections, manual annotation of tomographic data has been an important method to help understand the overall 3D morphology of cellular compartments. Here, we postulate that template matching can provide a tool for more objective annotation and contouring of cellular structures. Also, this technique can extract information hitherto unharvested in tomographic studies. To evaluate the performance of template matching on tomograms of stained sections, we generated several templates representing a piece of microtubule or patches of membranes of different staining-thicknesses. These templates were matched to tomograms of stained electron microscopy sections. Both microtubules and ER-Golgi membranes could be detected using this method. By matching cuboids of different thicknesses, we were able to distinguish between coated and non-coated endosomal membrane-domains. Finally, heterogeneity in staining-thickness of endosomes could be observed. Template matching can be a useful addition to existing annotation-methods, and provide additional insights in cellular architecture. © 2006 Elsevier Inc. All rights reserved.

The Focused Ion Beam – Scanning Electron Microscope (FIB-SEM) is a versatile instrument originating from the semiconductor industry. The FIB is used to produce cross sections of pre-defined locations of interest, which are imaged and analyzed with the SEM. Repeated FIB cross sectioning and subsequent SEM imaging –a process called FIB-SEM tomography– results in a full three-dimensional analysis of the structure of interest, allowing for the 3D reconstruction of cellular materials or porous media. In recent years, the capabilities of the FIB-SEM have attracted a considerable amount of attention from various scientific disciplines. This PhD Thesis presents a survey across samples from Life Sciences, Earth Sciences and Material Sciences, exploring and validating the application of the FIB-SEM instrument to scientific questions from the three disciplines. The common denominator of the various samples under investigation is their electrically insulating nature, requiring additional care in dealing with the samples in the microscope. Life Sciences Electron Microscopy (EM) in Life Sciences can be split in room temperature (cells are dehydrated, optionally embedded) and cryo applications. A contribution is made in both fields. Room temperature FIB-SEM tomography is demonstrated on cells embedded in a resin. The cellular membranes are stained with heavy metals, providing sufficient contrast in Back Scattered Electron (BSE) mode. It is shown that the distribution of endoplasmic reticulum and the mitochondria can be reconstructed in three dimensions. As for cryo-EM, a major quest in Life Sciences is the preparation of artifact-free thick cryo-sections or lamellas (>200 nm) for cryo-Transmission Electron Microscopy (TEM) tomography. A dedicated workflow has been developed to produce such thick cryo-sections with the FIB, while is SEM is used to check the quality of the sections in-situ. Earth Sciences The importance of 3D reconstructions over 2D imaging is demonstrated with an olivine sample. The permeability of olivine is linked to the wetting angle –the dihedral angle– of the melt and the rock. FIB-SEM tomography data of melt pockets indicates that measuring the dihedral angle based on 2D data may not be valid because of the presence of facets in the third dimension. Material Sciences Two different Material Sciences samples are investigated: 1) Large steamed zeolite ZSM-5 crystals and 2) Fluid Catalytic Cracking (FCC) particles. The steaming process of large ZSM-5 crystals creates mesoporosity, which is characterized by FIB-SEM tomography. To prevent charging artifacts, low-kV BSE imaging is used successfully. FIB-SEM tomography through the FCC particles revealed two major challenges considering the post-processing of the 3D data. The first challenge considers automated segmentation of the pore space from FIB-SEM tomography data. A combination of several techniques is found to be sufficiently accurate. The second challenge involves upscaling of transport properties from the dimensions analyzed by the FIB-SEM. A workflow is presented which employs experimentally based statistical means to extrapolate high resolution data to much larger length scales.

Autotransporters (ATs) are proteins secreted by Gram-negative bacteria that often play a role in virulence. Eight different ATs have been identified in Neisseria meningitidis, but only six of them have been characterized. AutA is one of the remaining ATs. Its expression remains controversial. Here, we show that the autA gene is present in many neisserial species, but its expression is often disrupted by various genetic features; however, it is expressed in certain strains of N. meningitidis. By sequencing the autA gene in large panels of disease isolates and Western blot analysis, we demonstrated that AutA expression is prone to phase variation at AAGC nucleotide repeats located within the DNA encoding the signal sequence. AutA is not secreted into the extracellular medium, but remains associated with the bacterial cell surface. We further demonstrate that AutA expression induces autoaggregation in a process that, dependent on the particular strain, may require extracellular DNA (eDNA). This property influences the organization of bacterial communities like lattices and biofilms. In vitro assays evidenced that AutA is a self-associating AT that binds DNA. We suggest that AutA-mediated autoaggregation might be particularly important for colonization and persistence of the pathogen in the nasopharynx of the host.

Human Immunodeficiency Virus type 1 (HIV-1) is one of the major health problems worldwide and has been for over thirty years. Most (67%) of the people infected with HIV-1 are living in sub-Saharan Africa. Here, the access to treatments is limited and most women are not in a position to protect themselves from getting infected. The aim of this study was to identify VHH that are both broad and potent HIV-1 neutralizers, which can be used in the fight against HIV-1. A VHH is an antibody fragment containing the antigen binding domain derived from a heavy chain only antibody. This special type of antibody can be found in members of the camel family. The advantage of VHH is that they are relatively small, stable, cheap to produce and easy to work with. In order to raise VHH against HIV-1, two llamas were immunized with a mix of its envelope proteins. After cloning of the llama’s VHH repertoire into a phagemid expression system, multiple different strategies were followed to identify neutralizing VHH. In total, twelve families of neutralizing VHH were found and characterized in more detail. These VHH target four independent areas on the HIV-1 envelope protein. The best VHH of them being J3, which neutralized 96 out of a 100 tested viruses with a median IC50 of 0.9 µg/ml and is targeting the CD4 binding site. The CD4 binding site on the envelope protein is considered an important area as the first interaction between virus and its target cell occurs via CD4. Previous studies have shown that linking two VHH together into so-called biheads may increase their breadth and potency. For this reason we have linked two different VHH, targeting independent epitopes, together to form bi-specific biheads. Several combinations of VHH have been tried, the best (J3 linked to 2E7) yielded upto 1400 times increase in potency compared to a mix of the individual VHH. Another bihead (J3 linked to 1F10) was capable of neutralizing a viral strain J3 alone could not neutralize, possibly improving the breadth of this molecule above 96%.

Alzheimer's Disease (AD) is the most common form of dementia among elderly in the Western world. AD is a devastating neurodegenerative disease where patients starting with episodic memory problems end up completely bedridden and care dependent. At present there is no real therapy stopping or reversing AD progression. Roughly 100 years ago Alois Alzheimer described the deposition of amyloid-beta (Aβ) in post-mortem brains, one of the hallmarks of AD. Since this description AD has had great attention. However, the exact mechanism of AD remains unclear, a general consensus is reached over Aβ as a peptide involved in AD progression. Aβ is derived from amyloid-beta precursor protein (APP) by the proteolytic cleavage activity of beta-secretase BACE1. Interesting, next to AD patients, healthy individuals also produce Aβ in their brain. So next to the production and presence of Aβ, an extra mechanism is in place to exert toxicity and therefore AD progression. The hydrophobic nature of Aβ makes this peptide prone to aggregate. Overwhelming evidence point to an aggregated form of Aβ (oligomer) as the culprit of toxicity. This makes aggregation of Aβ the mechanism responsible for the generation of toxic Aβ species. Members from the Camelidae family possess, next to conventional IgGs, a heavy chain only antibody. The antigen-binding domain of this antibody (VHH) is fully function while being about ten times smaller compared to a conventional antibody. Next to size, VHH have several other benefits like stability, production costs and genetic modulation. Next to that, VHH are found that inhibit protein aggregation and enzyme activity. We exploited the capability of VHH to inhibit protein aggregation by selecting and generation VHH against Aβ. Llamas were immunized with patient material containing severe Aβ deposition. The subsequently generated VHH library yielded one VHH that inhibits aggregation, and reduced Aβ toxicity. Mutation studies and an additional crystal structure led to the hypothesis that this VHH binds to a small aggregated form of Aβ. A secondary strategy to prevent Aβ toxicity is to inhibit Aβ production by targeting BACE1. We selected a VHH that inhibits BACE1 activity in vitro and in vivo. The in vivo pilot experiment showed a reduced concentration of Aβ and therefore might be of therapeutical value. Problematic is the delivery of compounds to the brain due to the blood brain barrier (BBB). We provided a proof of principle to deliver compounds over cell layers that were BBB models. Transferrin Receptor (TfR) was used to piggy bag an anti-TfR covalently coupled to an anti-Aβ over the cell layer. The aim of this thesis was to find VHH against Ab targets and characterize these VHH. We made great progress in characterizing VHH G7 in vitro and explain characteristics needed for aggregation and toxicity inhibition. Besides this we found a VHH inhibiting BACE1 activity in vitro and in vivo providing a secondary therapeutic strategy for AD. Furthermore, proof of principle is provided to deliver compounds over cellular monolayers modeling the blood brain barrier.

Delivery of drugs to sites of disease can be hampered by the presence of cellular barriers in the human body. The presence of such barriers reduces the therapeutic activity of drugs on the disease target. The barrier present between the bloodstream and the brain for instance, stringently controls what goes into the brain and what not. Diseases that take place in such enclosed systems may be difficult to reach for pharmaceutical compounds because of these barriers and this can reduce their therapeutic effectivity. In this thesis we have described the development of molecules that interact with specific barriers in the human body and that mediate translocation across these barriers. As platform we used antibodies from the llama. Llama antibodies have several favourable properties as compared with other mammalian antibodies. The llama antibody platform was used in the described studies to make entities capable of translocating across cellular barriers. Part of the described studies has looked at the removal of compounds from the body by efflux via the intestinal wall (a cellular barrier). We developed molecules which can interact specifically with that barrier and mediate the passage of unwanted molecules. These molecules can be used for example for the removal of viruses that have infected the body. Fusion of the translocation molecules with compounds that bind viruses would enable the resulting compounds to both bind the virus and mediate its translocation across the intestinal wall. Such therapeutic strategies could tackle the infections of persistent viruses such as HIV. The other part of the described studies has looked at the delivery of blood borne compounds across the blood brain barrier to the central nervous system. Efficient platforms that mediate brain delivery from the bloodstream are scarce/ non-existent. We found molecules which have favourable properties in artificial model systems mimicking the blood-brain barrier. Application of these molecules to mice showed that there was a significant proportion of the injected dose able to accumulate in the brain, which indicates that our molecules indeed can target the brain. Further genetic adaptation of the antibodies might improve the translocation process to the brain environment and deliver a bigger pool of therapeutics to the site of disease. Development of these molecules might yield entities capable of delivering therapeutically relevant compounds to the brain. At the moment, brain delivery of pharmaceuticals is still a feature that is hardly possible without invasive procedures. Due to this aberrant delivery, there is no proper treatment possible of diseases like Alzheimer’s, Parkinson’s and brain cancer. We hope that with further development, our molecules will be able to give a solution to the brain delivery problem and thereby aid in the treatment of brain afflictions. This thesis has given examples of how antibodies from the llama can be used as retargeting agents. We have shown that they can be designed to translocate across cellular barriers and to take along cargo in this process. Development of the antibodies may yield molecules which can help in the diagnosis and treatment of several diseases.

One of the major challenges for correlative microscopy is the preparation of the sample; the protocols for transmission electron microscopy (TEM) and fluorescence microscopy (FM) often prove to be incompatible. Here, we introduce 2+Staining: an improved contrasting procedure for Tokuyasu sections that yields both excellent positive membrane contrast in the TEM and bright fluorescence of the probe labeled on the section. 2+Staining involves the contrasting of the immunolabeled sections with 1% osmium tetroxide, 2% uranyl acetate and lead citrate in sequential steps, followed by embedding in 1.8% methyl cellulose. In addition, we demonstrate an amplification of the fluorescent signal by introducing additional antibody incubation steps to the immunolabeling procedure. The methods were validated using the integrated laser and electron microscope (iLEM), a novel tool for correlative microscopy combining FM and TEM in a single setup. The approaches were tested on HL-60 cells labeled for lysosomal-associated membrane protein 2 (LAMP-2) and on sections of muscle from a facioscapulohumeral dystrophy mouse model. Yielding excellent results and greatly expediting the workflow, the methods are of great value for those working in the field of correlative microscopy and indispensible for future users of integrated correlative microscopy.

Binding of tissue-type plasminogen activator (tPA) to amyloid and denatured proteins is reported in a number of studies. The binding site has been mapped previously to the finger domain of tPA. In this study, tPA and truncated tPA constructs, lacking the finger domain, were tested for their ability to bind to Aβ and AIAPP amyloid-like fibrils. Surface plasmon resonance experiments and pull-down assays clearly show that indeed tPA binds, but that the finger domain is not essential. Another possible binding mechanism via the lysine binding site on the kringle 2 domain was also not crucial for the binding. Immuno-electron microscopy studies show that tPA binds to fibril sides. This study shows that, besides the finger domain, other domains in tPA are involved in amyloid binding.

A number of studies have shown that induction of pulmonary toxicity by nanoparticles of the same chemical composition depends on particle size, which is likely in part due to differences in lung deposition. Particle size mostly determines whether nanoparticles reach the alveoli, and where they might induce toxicity. For the risk assessment of nanomaterials, there is need for a suitable dose metric that accounts for differences in effects between different sized nanoparticles of the same chemical composition. The aim of the present study is to determine the most suitable dose metric to describe the effects of silver nanoparticles after short-term inhalation. Rats were exposed to different concentrations (ranging from 41 to 1105 µg silver/m3 air) of 18, 34, 60 and 160 nm silver particles for four consecutive days and sacrificed at 24 h and 7 days after exposure. We observed a concentration-dependent increase in pulmonary toxicity parameters like cell counts and pro-inflammatory cytokines in the bronchoalveolar lavage fluid. All results were analysed using the measured exposure concentrations in air, the measured internal dose in the lung and the estimated alveolar dose. In addition, we analysed the results based on mass, particle number and particle surface area. Our study indicates that using the particle surface area as a dose metric in the alveoli, the dose–response effects of the different silver particle sizes overlap for most pulmonary toxicity parameters. We conclude that the alveolar dose expressed as particle surface area is the most suitable dose metric to describe the toxicity of silver nanoparticles after inhalation.

Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a well-established imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution.

Heavy-chain antibodies are present in llamas next to conventional immunoglobulins. Their variable domain, also known as VHH is a small, single-chain globular protein with full antigen-binding capacity. Its single-domain nature and some structural differences from conventional immunoglobulins make it a tool with high potential for a number of biotechnological and medical applications. The HIV pandemic represents since three decades a dramatic health problem throughout the world and especially in resource-limited countries. As an alternative to vaccination, prevention is regarded as the best way to control the pandemic, both from a healthcare and economic point of view. The development of microbicides that help preventing sexually transmitted infections of HIV (the most common way the virus is transmitted) is therefore highly desirable. In this thesis, several aspects of the development of llama heavy-chain antibody fragments as active compound of topical microbicides are investigated. In chapter one the 3D structure of the HIV neutralizing VHH D7 was determined. The extension, flexibility and amino acid-composition of its CDRs were compared to other known (human) neutralizing antibodies in order to define the way D7 neutralizes the virus by binding gp120. Structural comparison suggested diverse modes of interaction. Mutational analysis identified CDR3 as key area of the interaction with gp120, whose conformational flexibility is likely to accommodate multiple modes of antibody binding inside the CD4 binding site. Comparison of the related VHH A12 and D7 revealed broad and narrow cross-clade neutralization phenotypes respectively. In chapter two a family-specific phage library was generated and a number of homologous VHH was selected with varying neutralizing profiles. Analysis of their sequences and their recognition of a panel of different HIV-1 subtypes, allowed the understanding of the molecular basis of the different neutralization behaviors. In particular the important role played by the last two C-terminal amino acids of CDR3 was demonstrated. Secretion efficiency of VHH is a particularly important factor when large amounts are needed at low production cost. In chapter three we analyzed databases of structures and sequences to find sequence-related factors that influence the yield of VHH when produced in the host S. cerevisiae. We showed how the combined presence of five key residues and specific J segments is fundamental for proper folding. We also suggested that interactions with ER-resident chaperones are necessary to achieve high secretion efficiency. In the last chapter, VHH A12 and D7 were used as models to study their availability, activity and stability in a VHH-based topical microbicide. We systematically analyzed likely challenges that VHH will have to face in case of use in sub-tropical settings of resource-limited countries, as HIV preventative drug. VHH proved to be readily available when formulated as tablets or aqueous gel and very stable after extended incubation at physiological temperature and at low pH. We also investigated the tissue-penetration kinetics of VHH in order to assess whether the drug would be present in the sub-mucosal layers of the vagina and once there, carry out its protective action.

The study of lipoproteins, natural nanoparticles comprised of lipids and apolipoproteins that transport fats throughout the body, is of key importance to better understand, treat, and prevent cardiovascular disease. In the current study, we have developed a lipoprotein-based nanoparticle that consists of a quantum dot (QD) core and Cy5.5 labeled lipidic coating. The methodology allows judicious tuning of the QD/Cy5.5 ratio, which enabled us to optimize Förster resonance energy transfer (FRET) between the QD core and the Cy5.5-labeled coating. This phenomenon allowed us to study lipoprotein-lipoprotein interactions, lipid exchange dynamics, and the influence of apolipoproteins on these processes. Moreover, we were able to study HDL-cell interactions and exploit FRET to visualize HDL association with live macrophage cells

An apparatus for observing a sample (1) with a TEM column and an optical high resolution scanning microscope (10). The sample position when observing the sample with the TEM column differs from the sample position when observing the sample with the optical microscope in that in the latter case the sample is tilted towards the light-optical microscope. By using an optical microscope of the scanning type, and preferably using monochromatic light, the lens elements (11) of the optical microscope facing the sample position can be sufficiently small to be positioned between the pole faces (8A, 8B) of the (magnetic) particle-optical objective lens (7). This is in contrast with the objective lens systems conventionally used in optical microscopes, which show a large diameter. Furthermore the optical microscope, or at least the parts (11) close to the sample, may be retractable so as to free space when imaging in TEM mode

Starless molecular sieves: The starlike appearence of confocal fluorescence microscopy (CFM) images of large crystals of the AlPO-5 family of microporous materials is due to the presence of barriers to molecular diffusion in the internal crystal architecture (see picture) rather than a star-shaped subcrystal according to studies by CFM, focused ion beam milling, electron backscatter diffraction, and atomic force microscopy.

The current activation model of the EGF receptor (EGFR) predicts that binding of EGF results in dimerization and oligomerization of the EGFR, leading to the allosteric activation of the intracellular tyrosine kinase. Little is known about the regulatory mechanism of receptor oligomerization. In this study, we have employed FRET between identical fluorophores (homo-FRET) to monitor the dimerization and oligomerization state of the EGFR before and after receptor activation. Our data show that, in the absence of ligand, ∼40% of the EGFR molecules were present as inactive dimers or predimers. The monomer/predimer ratio was not affected by deletion of the intracellular domain. Ligand binding induced the formation of receptor oligomers, which were found in both the plasma membrane and intracellular structures. Ligand-induced oligomerization required tyrosine kinase activity and nine different tyrosine kinase substrate residues. This indicates that the binding of signaling molecules to activated EGFRs results in EGFR oligomerization. Induction of EGFR predimers or pre-oligomers using the EGFR fused to the FK506-binding protein did not affect signaling but was found to enhance EGF-induced receptor internalization. Our data show that EGFR oligomerization is the result of EGFR signaling and enhances EGFR internalization

Cells are organized in a highly complex manner. And while there are many different types of cells - each organized in a different manner according to their function - they do share certain commonalities. Among these commonalities are membranes that functions not only as a barrier between the extra- and intra-cellular environment, but is also involved in many cell-biological processes like signaling and transport; and since each of these processes require a different set of components and organization, a large heterogeneity is expected within these membranes. Electron microscopic techniques have been invaluable to our understanding of the complex cellular architecture (and the corresponding dynamics). In recent years, 3D electron microscopic techniques like electron tomography and the focussed ion-beam scanning electron microscope have emerged and provided us with an exciting new perspective of cellular structures and their inter- and intra-organellar relationships. However, extracting relevant information from (3D) micrographs heavily relies on manual segmentation techniques and is therefore affected by the eye of the beholder. In the work presented in this thesis we have explored template matching as an approach to harvest biologically relevant information from tomographic volumes in a (semi-)objective manner. By using a stylized cuboid-shaped template we could extract membranes from the tomographic volumes and thus study the overall morphology of organelles. But more importantly, by comparing the matching results of slightly distinct templates, we were able to visualize ultra-structural details that are lost by manual segmentation. By applying this approach we up the ante of electron tomographic approaches and were able to bring it into a new realm where we can study and visualize in three dimensions the membrane-organization of biologically functional and significant events like lipid- and protein-domains.

The full potential in catalyst development will only be realized if characterization techniques are available that can probe materials with subnanometer resolution. One of the most employed techniques to image heterogeneous catalysts at the nanometer and subnanometer scale is transmission electron microscopy (TEM). As suggested by the name, TEM uses electrons transmitted through the object for imaging. Since the interaction between electrons and matter is very strong, only thin parts, commonly much less than a micron in thickness, are imaged. Since heterogeneous catalysts are, in most cases, structured on a much smaller length scale, the sample thickness can be reduced to TEM requirements by appropriate preparation techniques and is, therefore, no limitation.

In this paper, we present a comprehensive study of the sol-gel transitions and liquid crystal phase transitions in aqueous suspensions of positively charged colloidal gibbsite platelets at pH 4-5 over a wide range of particle concentrations (50-600 g/L) and salt concentrations (10-4-10-1 M NaCl). A detailed sol-gel diagram was established by oscillatory rheological experiments. These demonstrate the presence of kinetically arrested states both at high and at low salt concentrations, enclosing a sol region. Birefringence and iridescence show that in the sol state nematic and hexagonal columnar liquid crystal phases are formed. The gel and liquid crystal structures are studied in further detail using small-angle X-ray scattering (SAXS) and cryo-focused ion beam/scanning electron microscopy (cryo-FIB-SEM). The gel formed at high salt concentration shows signatures of a sponge-like structure and does not display birefringence. In the sol region, by lowering the salt concentration and/or increasing the gibbsite concentration, the nematic phase gradually transforms from the discotic nematic (ND) into the columnar nematic (NC) with much stronger side-to-side interparticle correlations. Subsequently, this NC structure can be either transformed into the hexagonal columnar phase or arrested into a birefringent repulsive gel state with NC structure.

We report an experimental and microstructural study of the frictional properties of simulated fault gouges prepared from natural limestone (96 % CaCO3) and pure calcite. Our experiments consisted of direct shear tests performed, under dry and wet conditions, at an effective normal stress of 50 MPa, at 18–150 °C and sliding velocities of 0.1–10 μm/s. Wet experiments used a pore water pressure of 10 MPa. Wet gouges typically showed a lower steady-state frictional strength (μ = 0.6) than dry gouges (μ = 0.7–0.8), particularly in the case of the pure calcite samples. All runs showed a transition from stable velocity strengthening to (potentially) unstable velocity weakening slip above 80–100 °C. All recovered samples showed patchy, mirror-like surfaces marking boundary shear planes. Optical study of sections cut normal to the shear plane and parallel to the shear direction showed both boundary and inclined shear bands, characterized by extreme grain comminution and a crystallographic preferred orientation. Cross-sections of boundary shears, cut normal to the shear direction using focused ion beam—SEM, from pure calcite gouges sheared at 18 and 150 °C, revealed dense arrays of rounded, ~0.3 μm-sized particles in the shear band core. Transmission electron microscopy showed that these particles consist of 5–20 nm sized calcite nanocrystals. All samples showed evidence for cataclasis and crystal plasticity. Comparing our results with previous models for gouge friction, we suggest that frictional behaviour was controlled by competition between crystal plastic and granular flow processes active in the shear bands, with water facilitating pressure solution, subcritical cracking and intergranular lubrication. Our data have important implications for the depth of the seismogenic zone in tectonically active limestone terrains. Contrary to recent claims, our data also demonstrate that nanocrystalline mirror-like slip surfaces in calcite(-rich) faults are not necessarily indicative of seismic slip rates.

BACKGROUND:: Risk prediction of rupture of intracranial aneurysms is poor, and mainly based on lumen characteristics. However, characteristics of the aneurysm wall may be more informative predictors. The limited resolution of currently available imaging techniques, and the thin aneurysm wall, make imaging of wall thickness challenging. OBJECTIVE:: To introduce a novel protocol for imaging wall thickness variation using ultra-high resolution 7.0 Tesla MRI. METHODS:: We studied 33 unruptured intracranial aneurysms in 24 patients with a T1-weighted three-dimensional magnetization prepared inversion recovery turbo-spin-echo whole brain sequence with a resolution of 0.8 mm x 0.8 mm x 0.8 mm. We performed a validation study with a wedge phantom, and with two aneurysm wall biopsies obtained during aneurysm treatment, using ex vivo MRI and histological examination, and correlating variations in MRI signal intensity with variations in actual thickness of the aneurysm wall. RESULTS:: In vivo, the aneurysm wall was visible in 28 of the 33 aneurysms. Variation in signal intensity was observed in all visible aneurysm walls. Ex vivo MRI showed variation in signal intensity across the wall of the biopsies, similar as observed on the in vivo images. Signal intensity and actual thickness in both biopsies had a linear correlation with Pearson correlation coefficients of 0.85 and 0.86. CONCLUSION:: Unruptured intracranial aneurysm wall and its variation in thickness can be visualized with 7.0 Tesla MRI. Aneurysm wall thickness variation can now be further studied as a risk factor for rupture in prospective studies.

Lipids are essential for cellular life, functioning either organized as bilayer membranes to compartmentalize cellular processes, as signaling molecules or as metabolic energy storage. Our current knowledge on lipid organization and cellular lipid homeostasis is mainly based on biochemical data. However, the resolving power of the most commonly used biochemical methods (molecular analysis of cellular fractions and the use of fluorescent lipid analogs) is limited. To gain further insight in subcellular lipid distribution and processes involved in cellular lipid homeostasis requires a high resolution method, allowing the study of lipids within the cellular context. In this thesis, we explored the use of one such method, transmission electron microscopy, to study cellular lipids, lipid droplets and lipoproteins at the ultrastructural level. Preparation of biological samples for electron microscopy (EM) is aimed at either immobilizing or removing water within the sample while retaining the ultrastructural organization of the cell. Based on the existing literature, lipid preservation in EM sample preparation was examined. From this, parameters determining lipid extraction were derived, allowing us to assess lipid retention in a given sample preparation procedure. This information was applied in studying approaches to visualize the biogenesis of lipid droplets (LDs). Using several preparation- and imaging techniques, we found indications that LD biogenesis occurs in the mitochondria-associated ER membranes, making critical progress in visualizing the initial stages of lipid droplet formation. Interaction between LDs and other cellular organelles was assessed by reconstructing 3d volumes from recorded tilt-series, allowing us to show direct membrane contacts between LDs and ER membrane and between an LD and a lysosome. The formation of very low density lipoproteins (VLDL), the main carriers of neutral lipid in the blood circulation, was investigated. Immuno-gold labeling on ApoB, the main apo-protein on VLDL, could in principle be used to study the synthesis route of VLDL. However, the current 'on-section’-labeling strategies often suffer from a loss of antigenicity of ApoB. We show that this is most likely due to delipidation of ApoB by the lipid scavenging action of bovine serum albumin, used as a blocking agent during the labeling procedure. By reducing de-lipidation, we are able to successfully localize ApoB by immuno-gold labeling. We were able to trace the flow of de-novo synthesized cellular lipid at the ultrastructural level, using a clickable fatty acid, combined with an optimized specimen preparation procedure. This lipid flow could be changed and re-directed using specific inhibitors. Changes were visualized within the cell by immuno-gold labeling, validating the use of clickable fatty acids as a suitable tool for imaging lipid flows on the nanometer scale. Lastly we present a novel method for high-pressure freezing and frozen hydrated sectioning of cells attached to the substrate they were grown on, providing a less artifact-prone method for frozen hydrated sectioning and cryo-electron microscopy of vitreous sections (CEMOVIS). This method reduces sample preparation time, allows the use of shorter incubation times and brings electron microscopy one step closer to the ultimate goal: visualizing cells in their native state at high resolution.

In this paper, lithographic methods are successfully employed to create growth templates for colloidal self-assembly, enabling the inclusion of crystallographic defects at predetermined positions. It is shown that through smart template design stacking faults can be grown predictably into face centered cubic structures. More interestingly, by precise guiding of the stacking faults hollow intergrowth channels can be grown at predetermined lateral and vertical positions. The mechanisms involved in defect growth are promising for extension of this technique to more complex crystal structures, such as the diamond structure, as well as to more complex faults, including corners and t-junctions.

Protein kinase C epsilon (PKCε), which is one of the novel PKC isozymes, is widely expressed throughout the body and has important roles in the function of the nervous, cardiovascular and immune systems. In order to better understand PKCε regulated pathways, isozyme specific activity modulators are desperately needed. Such compounds could also be developed into drugs for diseases such as diabetes, cancer and Alzheimer’s disease, where PKCε dysregulation is implicated. This thesis describes the selection and characterization of PKCε specific llama single chain antibodies, known as VHHs. VHHs have several advantages compared to the conventional antibodies consisting of two heavy and two light chains. VHHs are small, easy to clone, convenient to genetically modify, remarkably stable, and tend to be highly specific for the target antigen. PKCε specific VHHs were selected in one round from an immune phage-VHH library using phage display. The VHHs that showed the strongest binding to human PKCε were produced in E. coli as monovalent proteins and tested for their ability to influence PKCε activity in in vitro kinase activity assays. Three VHHs (A10, C1 and D1) were found to increase the activity of PKCε, whereas three other VHHs (C7, E6 and G8) inhibited PKCε kinase activity. These effects were PKCε specific, since the VHHs had no effect on the activity of PKCδ, PKCθ or protein kinase A. A more detailed kinetic analysis revealed that the activators increased PKCε activity by increasing the maximum rate of the reaction and/or by speeding up the reaction. Furthermore, VHHs E6 and G8 were found to be non-competitive enzyme inhibitors. Interestingly, the binding site of all of the tested VHHs was in the catalytic domain of PKCε. Moreover, the binding of the VHHs was found to be conformation dependent and species specific. In order to study whether the VHHs could also influence PKCε activity in a cellular context, the VHHs were cloned into a mammalian expression vector with a C-terminal mCherry tag, and the VHH-mCherry proteins were transiently expressed in HeLa cells. Several cellular effects, such as changes in cell morphology, increases in cytotoxicity, and inhibition of PKCε downregulation, were observed for some of the VHH-mCherry constructs. The most direct indication that the VHHs can also influence PKCε activity in cells came from PKCε-EGFP translocation studies, where the activator A10-mCherry increased the rate of PKCε-EGFP translocation to the membranes in response to phorbol ester stimulation. In contrast, the inhibitors C7-mCherry and G8-mCherry decreased the rate of translocation. The results presented in this thesis demonstrate that highly specific VHHs against an intracellular antigen can be selected in one round from an immune phage-VHH library. Furthermore, it was shown that the selected VHHs can act as activators or inhibitors of PKCε activity in vitro and in cells. In addition to the potential therapeutic applications of the VHHs for diseases with aberrant PKCε signalling, these PKCε specific activity modulators will be valuable tools in research into PKCε regulated pathways

Diagnosis of the hepatitis B Virus (HBV) is important for treatment and prevention of further spread of the virus. Today, detection of hepatitis B virus surface antigen (HBsAg) is the method of choice for the screening and initial diagnosis of HBV. Genetic diversity and discovery of HBV variants with mutations in the immunological dominant region of HBsAg caused none reactivity in some diagnostic assays. Characterization of anti-HBs monoclonal antibodies lead to discovery of a unique monoclonal antibody revealing the true topology of the small s-protein. The knowledge of the antibody recognition spectra, lead to the development of a diagnostics assay showing for the first time detection of all HBsAg mutants based on three different epitope regions. The three epitope regions should in theory enable detection of all known HBsAg mutants, which was proven after testing HBsAg mutants in direct comparison to other diagnostic assay lacking complete mutant detection. The improved HBsAg screening assay permitted better and earlier diagnosis of HBV infections, leaving the diagnosed HBV patient for treatment. Today the main motivation for intervening in an on-going HBV infection is in case the liver is compromised. Depending on the status of the patient, antiviral therapy is started or in worse case when the liver stops to function, a liver transplantation is the final choice. Transplantation of the liver is preceded by antiviral therapy in combination with Hepatitis B immunoglobulin (HBIg) additions. In a quest for an alternative source for HBig we isolated and selected single-domain antibodies (VHHs) that recognize the major small s-protein of HBV. Testing of five VHH in an in-vitro neutralization experiment identified one VHH that could neutralize HBV comparable with good neutralizing anti-HBs reference antibodies. Pepscan analysis and amino acid substitution experiments suggested mutual recognition of the VHH of both the “a”-determinant and c-terminus of the s-protein, fixating the structure and preventing conformational changes needed during viral entry. Alternatively binding to a yet to be discovered HBV (co-) receptor is blocked by the VHH. Ab initio modeling of the dimer “a”-determinant sequence (aa100-155) with VHH S5 revealed a tempting structure that could explain our and published observations. Mainly the location of the cysteine residues in the newly found structure was striking, showing possible formation of inter and intra chain disulfide bridges. Separate analysed c-terminal hydrophobic region (155-226) showed structural homology with Arfaptin 2, a BAR domain protein. The homologous structure predicted importance of the region giving curvature to the aggregated s-proteins in the HBsAg particles. We conclude from the modelling data that the protruding spikes are dimers of the “a”-determinant with peripheral stabilized c-terminal s-protein fragments on the 3- and 5-fold axis.

Homo-FRET, Forster resonance energy transfer between identical fluorophores, can be conveniently measured by observing its effect on the fluorescence anisotropy. This review aims to summarize the possibilities of fluorescence anisotropy imaging techniques to investigate clustering of identical proteins and lipids. Homo-FRET imaging has the ability to determine distances between fluorophores. In addition it can be employed to quantify cluster sizes as well as cluster size distributions. The interpretation of homo-FRET signals is complicated by the fact that both the mutual orientations of the fluorophores and the number of fluorophores per cluster affect the fluorescence anisotropy in a similar way. The properties of the fluorescence probes are very important. Taking these properties into account is critical for the correct interpretation of homo-FRET signals in protein- and lipid-clustering studies. This is be exemplified by studies on the clustering of the lipid raft markers GPI and K-ras, as well as for EGF receptor clustering in the plasma membrane.

Binary nanocrystal superlattices, that is, ordered structures of two sorts of nanocolloids, hold promise for a series of functional materials with novel collective properties. Here we show that based on electron tomography a comprehensive, quantitative, three-dimensional characterization of these systems down to the single nanocrystal level can be achieved, which is key in understanding the emerging materials properties. On four binary lattices composed of PbSe, CdSe, and Au nanocrystals, we illustrate that ambiguous interpretations based on two-dimensional transmission electron microscopy can be prevented, nanocrystal sizes and superlattice parameters accurately determined, individual crystallographic point and plane defects studied, and the order/disorder at the top and bottom surfaces imaged. Furthermore, our results suggest that superlattice nucleation and growth occurred at the suspension/air interface and that the unit cells of some lattices are anisotropically deformed upon drying.

Correlative fluorescence and electron microscopy has become an indispensible tool for research in cell biology. The integrated Laser and Electron Microscope (iLEM) combines a Fluorescence Microscope (FM) and a Transmission Electron Microscope (TEM) within one set-up. This unique imaging tool allows for rapid identification of a region of interest with the FM, and subsequent high resolution TEM imaging of this area. Sample preparation is one of the major challenges in correlative microscopy of a single specimen; it needs to be apt for both FM and TEM imaging. For iLEM, the performance of the fluorescent probe should not be impaired by the vacuum of the TEM. In this technical note, we have compared the fluorescence intensity of six fluorescent probes in a dry, oxygen free environment relative to their performance in water. We demonstrate that the intensity of some fluorophores is strongly influenced by its surroundings, which should be taken into account in the design of the experiment. Furthermore, a freeze-substitution and Lowicryl resin embedding protocol is described that yields excellent membrane contrast in the TEM but prevents quenching of the fluorescent immuno-labeling. The embedding protocol results in a single specimen preparation procedure that performs well in both FM and TEM. Such procedures are not only essential for the iLEM, but also of great value to other correlative microscopy approaches.

Staphylococcus aureus is a versatile pathogen capable of causing a broad range of diseases in many different hosts. S. aureus can adapt to its host through modification of its genome (e.g. by acquisition and exchange of mobile genetic elements that encode host-specific virulence factors). Recently, the prophage φSaeq1 was discovered in S. aureus strains from six different clonal lineages almost exclusively isolated from equids. Within this phage, we discovered a novel variant of staphylococcal complement inhibitor (SCIN), a secreted protein that interferes with activation of the human complement system, an important line of host defense. We here show that this equine variant of SCIN, eqSCIN, is a potent blocker of equine complement system activation and subsequent phagocytosis of bacteria by phagocytes. Mechanistic studies indicate that eqSCIN blocks equine complement activation by specific inhibition of the C3 convertase enzyme (C3bBb). Whereas SCIN-A from human S. aureus isolates exclusively inhibits human complement, eqSCIN represents the first animal-adapted SCIN variant that functions in a broader range of hosts (horses, humans, and pigs). Binding analyses suggest that the human-specific activity of SCIN-A is related to amino acid differences on both sides of the SCIN-C3b interface. These data suggest that modification of this phage-encoded complement inhibitor plays a role in the host adaptation of S. aureus and are important to understand how this pathogen transfers between different hosts.

For toxicity testing of airborne particles, air-liquid interface (ALI) exposure systems have been developed for in vitro tests in order to mimic realistic exposure conditions. This puts specific demands on the cell culture models. Many cell types are negatively affected by exposure to air (e.g., drying out) and only remain viable for a few days. This limits the exposure conditions that can be used in these models: usually relatively high concentrations are applied as a cloud (i.e., droplets containing particles, which settle down rapidly) within a short period of time. Such experimental conditions do not reflect realistic long-term exposure to low concentrations of particles. To overcome these limitations the use of a human bronchial epithelial cell line, Calu-3 was investigated. These cells can be cultured at ALI conditions for several weeks while retaining a healthy morphology and a stable monolayer with tight junctions. In addition, this bronchial model is suitable for testing the effects of repeated exposures to low, realistic concentrations of airborne particles using an ALI exposure system. This system uses a continuous airflow in contrast to other ALI exposure systems that use a single nebulization producing a cloud. Therefore, the continuous flow system is suitable for repeated and prolonged exposure to airborne particles while continuously monitoring the particle characteristics, exposure concentration, and delivered dose. Taken together, this bronchial model, in combination with the continuous flow exposure system, is able to mimic realistic, repeated inhalation exposure conditions that can be used for toxicity testing.

Background Although silver nanoparticles are currently used in more than 400 consumer products, it is not clear to what extent they induce adverse effects after inhalation during production and use. In this study, we determined the lung burden, tissue distribution, and the induction and recovery of adverse effects after short-term inhalation exposure to 15 nm and 410 nm silver nanoparticles. Methods Rats were nose-only exposed to clean air, 15 nm silver nanoparticles (179 μg/m3) or 410 nm silver particles (167 μg/m3) 6 hours per day, for four consecutive days. Tissue distribution and the induction of pulmonary toxicity were determined at 24 hours and 7 days after exposure and compared with the internal alveolar dose. Presence of silver nanoparticles in lung cells was visualized by transmission electron microscopy (TEM). Results Exposure to 15 nm silver nanoparticles induced moderate pulmonary toxicity compared to the controls, indicated by a 175-fold increased influx of neutrophils in the lungs, a doubling of cellular damage markers in the lungs, a 5-fold increase in pro-inflammatory cytokines, and a 1.5-fold increase in total glutathione at 24 hours after exposure. All the observed effects disappeared at 7 days after exposure. No effects were observed after exposure to 410 nm silver particles. The internal alveolar mass dose of the 15 nm nanoparticles was 3.5 times higher compared to the 410 nm particles, which equals to a 66,000 times higher particle number. TEM analysis revealed 15 nm nanoparticles in vesicles and nuclei of lung cells, which were decreased in size to

Human Immunodeficiency Virus type 1 (HIV-1) is one of the major health problems worldwide and has been for over thirty years. Most (67%) of the people infected with HIV-1 are living in sub-Saharan Africa. Here, the access to treatments is limited and most women are not in a position to protect themselves from getting infected. The aim of this study was to identify VHH that are both broad and potent HIV-1 neutralizers, which can be used in the fight against HIV-1. A VHH is an antibody fragment containing the antigen binding domain derived from a heavy chain only antibody. This special type of antibody can be found in members of the camel family. The advantage of VHH is that they are relatively small, stable, cheap to produce and easy to work with. In order to raise VHH against HIV-1, two llamas were immunized with a mix of its envelope proteins. After cloning of the llama’s VHH repertoire into a phagemid expression system, multiple different strategies were followed to identify neutralizing VHH. In total, twelve families of neutralizing VHH were found and characterized in more detail. These VHH target four independent areas on the HIV-1 envelope protein. The best VHH of them being J3, which neutralized 96 out of a 100 tested viruses with a median IC50 of 0.9 µg/ml and is targeting the CD4 binding site. The CD4 binding site on the envelope protein is considered an important area as the first interaction between virus and its target cell occurs via CD4. Previous studies have shown that linking two VHH together into so-called biheads may increase their breadth and potency. For this reason we have linked two different VHH, targeting independent epitopes, together to form bi-specific biheads. Several combinations of VHH have been tried, the best (J3 linked to 2E7) yielded upto 1400 times increase in potency compared to a mix of the individual VHH. Another bihead (J3 linked to 1F10) was capable of neutralizing a viral strain J3 alone could not neutralize, possibly improving the breadth of this molecule above 96%.

Alzheimer's Disease (AD) is the most common form of dementia among elderly in the Western world. AD is a devastating neurodegenerative disease where patients starting with episodic memory problems end up completely bedridden and care dependent. At present there is no real therapy stopping or reversing AD progression. Roughly 100 years ago Alois Alzheimer described the deposition of amyloid-beta (Aβ) in post-mortem brains, one of the hallmarks of AD. Since this description AD has had great attention. However, the exact mechanism of AD remains unclear, a general consensus is reached over Aβ as a peptide involved in AD progression. Aβ is derived from amyloid-beta precursor protein (APP) by the proteolytic cleavage activity of beta-secretase BACE1. Interesting, next to AD patients, healthy individuals also produce Aβ in their brain. So next to the production and presence of Aβ, an extra mechanism is in place to exert toxicity and therefore AD progression. The hydrophobic nature of Aβ makes this peptide prone to aggregate. Overwhelming evidence point to an aggregated form of Aβ (oligomer) as the culprit of toxicity. This makes aggregation of Aβ the mechanism responsible for the generation of toxic Aβ species. Members from the Camelidae family possess, next to conventional IgGs, a heavy chain only antibody. The antigen-binding domain of this antibody (VHH) is fully function while being about ten times smaller compared to a conventional antibody. Next to size, VHH have several other benefits like stability, production costs and genetic modulation. Next to that, VHH are found that inhibit protein aggregation and enzyme activity. We exploited the capability of VHH to inhibit protein aggregation by selecting and generation VHH against Aβ. Llamas were immunized with patient material containing severe Aβ deposition. The subsequently generated VHH library yielded one VHH that inhibits aggregation, and reduced Aβ toxicity. Mutation studies and an additional crystal structure led to the hypothesis that this VHH binds to a small aggregated form of Aβ. A secondary strategy to prevent Aβ toxicity is to inhibit Aβ production by targeting BACE1. We selected a VHH that inhibits BACE1 activity in vitro and in vivo. The in vivo pilot experiment showed a reduced concentration of Aβ and therefore might be of therapeutical value. Problematic is the delivery of compounds to the brain due to the blood brain barrier (BBB). We provided a proof of principle to deliver compounds over cell layers that were BBB models. Transferrin Receptor (TfR) was used to piggy bag an anti-TfR covalently coupled to an anti-Aβ over the cell layer. The aim of this thesis was to find VHH against Ab targets and characterize these VHH. We made great progress in characterizing VHH G7 in vitro and explain characteristics needed for aggregation and toxicity inhibition. Besides this we found a VHH inhibiting BACE1 activity in vitro and in vivo providing a secondary therapeutic strategy for AD. Furthermore, proof of principle is provided to deliver compounds over cellular monolayers modeling the blood brain barrier.

Delivery of drugs to sites of disease can be hampered by the presence of cellular barriers in the human body. The presence of such barriers reduces the therapeutic activity of drugs on the disease target. The barrier present between the bloodstream and the brain for instance, stringently controls what goes into the brain and what not. Diseases that take place in such enclosed systems may be difficult to reach for pharmaceutical compounds because of these barriers and this can reduce their therapeutic effectivity. In this thesis we have described the development of molecules that interact with specific barriers in the human body and that mediate translocation across these barriers. As platform we used antibodies from the llama. Llama antibodies have several favourable properties as compared with other mammalian antibodies. The llama antibody platform was used in the described studies to make entities capable of translocating across cellular barriers. Part of the described studies has looked at the removal of compounds from the body by efflux via the intestinal wall (a cellular barrier). We developed molecules which can interact specifically with that barrier and mediate the passage of unwanted molecules. These molecules can be used for example for the removal of viruses that have infected the body. Fusion of the translocation molecules with compounds that bind viruses would enable the resulting compounds to both bind the virus and mediate its translocation across the intestinal wall. Such therapeutic strategies could tackle the infections of persistent viruses such as HIV. The other part of the described studies has looked at the delivery of blood borne compounds across the blood brain barrier to the central nervous system. Efficient platforms that mediate brain delivery from the bloodstream are scarce/ non-existent. We found molecules which have favourable properties in artificial model systems mimicking the blood-brain barrier. Application of these molecules to mice showed that there was a significant proportion of the injected dose able to accumulate in the brain, which indicates that our molecules indeed can target the brain. Further genetic adaptation of the antibodies might improve the translocation process to the brain environment and deliver a bigger pool of therapeutics to the site of disease. Development of these molecules might yield entities capable of delivering therapeutically relevant compounds to the brain. At the moment, brain delivery of pharmaceuticals is still a feature that is hardly possible without invasive procedures. Due to this aberrant delivery, there is no proper treatment possible of diseases like Alzheimer’s, Parkinson’s and brain cancer. We hope that with further development, our molecules will be able to give a solution to the brain delivery problem and thereby aid in the treatment of brain afflictions. This thesis has given examples of how antibodies from the llama can be used as retargeting agents. We have shown that they can be designed to translocate across cellular barriers and to take along cargo in this process. Development of the antibodies may yield molecules which can help in the diagnosis and treatment of several diseases.

One of the major challenges for correlative microscopy is the preparation of the sample; the protocols for transmission electron microscopy (TEM) and fluorescence microscopy (FM) often prove to be incompatible. Here, we introduce 2+Staining: an improved contrasting procedure for Tokuyasu sections that yields both excellent positive membrane contrast in the TEM and bright fluorescence of the probe labeled on the section. 2+Staining involves the contrasting of the immunolabeled sections with 1% osmium tetroxide, 2% uranyl acetate and lead citrate in sequential steps, followed by embedding in 1.8% methyl cellulose. In addition, we demonstrate an amplification of the fluorescent signal by introducing additional antibody incubation steps to the immunolabeling procedure. The methods were validated using the integrated laser and electron microscope (iLEM), a novel tool for correlative microscopy combining FM and TEM in a single setup. The approaches were tested on HL-60 cells labeled for lysosomal-associated membrane protein 2 (LAMP-2) and on sections of muscle from a facioscapulohumeral dystrophy mouse model. Yielding excellent results and greatly expediting the workflow, the methods are of great value for those working in the field of correlative microscopy and indispensible for future users of integrated correlative microscopy.

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.