<p> </p><p><em>Trypanosoma theileri</em>, of the subgenus <em>Megatrypanum, </em>a non-pathogenic cosmopolitan blood dwelling parasite of bovine. <em>T. theileri </em>can be cultured at room temperature in several culture media.</p><p>Blood samples were collected from deer's. To see if the blood was infected with trypanosomes it was cultivated in 2 ml sheep blood or cell cultivation medium DMEM with antibiotics.</p><p>Growth was detected by microscopy to see if there were any trypanosomes.</p><p>To determine the species of trypanosomes that was in the deer blood a DNA-preparation was done before a Polymerase Chain Reaction (PCR) could be done. With sequencing the trypanosomes where determined to be<em> Trypanosoma theileri</em>.</p><p>Different tests were made to see in what way the trypanosomes best were caught to the objective slides.</p><p>Forty samples of borrelia positive serum from forty different patients were tested with the fluorescent microscopy. Forty different samples from blood donors were tested the same way.</p><p>Blood samples from 16 different fissiped were taken and to see if they were infected with trypanosomes. Three different PCR's were done on the 16 blood samples.</p><p>A small test on human blood was also performed.</p><p>Protein identification by immunoblot with western blot and silver staining was done.</p><p>With the electron microscopy tests were done in the ordinary way and Critical Dry Point to see if both of the techniques worked.</p><p>Enzyme-Linked Immuno Sorbent Assay (ELISA) test were accomplished on two 96 well plates. The wells on the plates were diluted in different ways before they were processed.</p><p> </p><p> </p>

Trypanosoma theileri, of the subgenus Megatrypanum, a non-pathogenic cosmopolitan blood dwelling parasite of bovine. T. theileri can be cultured at room temperature in several culture media. Blood samples were collected from deer's. To see if the blood was infected with trypanosomes it was cultivated in 2 ml sheep blood or cell cultivation medium DMEM with antibiotics. Growth was detected by microscopy to see if there were any trypanosomes. To determine the species of trypanosomes that was in the deer blood a DNA-preparation was done before a Polymerase Chain Reaction (PCR) could be done. With sequencing the trypanosomes where determined to be Trypanosoma theileri. Different tests were made to see in what way the trypanosomes best were caught to the objective slides. Forty samples of borrelia positive serum from forty different patients were tested with the fluorescent microscopy. Forty different samples from blood donors were tested the same way. Blood samples from 16 different fissiped were taken and to see if they were infected with trypanosomes. Three different PCR's were done on the 16 blood samples. A small test on human blood was also performed. Protein identification by immunoblot with western blot and silver staining was done. With the electron microscopy tests were done in the ordinary way and Critical Dry Point to see if both of the techniques worked. Enzyme-Linked Immuno Sorbent Assay (ELISA) test were accomplished on two 96 well plates. The wells on the plates were diluted in different ways before they were processed.

Our group has developed a family of single molecules termed nanocars, which are aimed at performing controllable motion on surfaces. In this work, a series of light-activated motorized nanomachines incorporated with a MHz frequency light-activated unidirectional rotary motor were designed and synthesized. We hope the light-activated motor can serve as the powering unit for the nanomachines, and perform controllable translational motion on surfaces or in solution. A series of motorized nanovehicles intended for scanning tunneling microscopy (STM) imaging were designed and synthesized. A p-carborane-wheeled motorized nanocar was synthesized and monitored by STM. Single-molecule imaging was accomplished on a Cu(111) surface. However, further manipulations did lead to motor induced lateral motion. We attributed this result to the strong molecule-surface interactions between the p-carborane-wheeled nanocar and the Cu(111) surface. To fine-tune the molecule-surface interactions, an adamantane-wheeled motorized nanocar and a three-wheel nanoroadster were designed and synthesized. In addition, the STM substrates will be varied and different combinations of molecule-surface interactions will be studied. As a complimentary imaging method to STM, single-molecule fluorescence microscopy (SMFM) also provides single-molecule level resolution. Unlike STM experiment requires ultra-high vacuum and conductive substrate, SMFM experiment is conducted at ambient conditions and uses non-conductive substrate. This imaging method allows us to study another category of molecule-surface interactions. We plan to design a fluorescent motorized nanocar that is suitable for SMFM studies. However, both the motor and fluorophore are photochemically active molecules. In proximity, some undesired energy transfer or interference could occur. A cyanine 5- (cy5-) tagged motorized nanocar incorporated with the MHz motor was designed and synthesized in order to minimize the potential energy transfer or interference between the motor and the fluorophore. The SMFM study of this cy5-tagged motorized nanocar is currently undergoing. The design of light-activated motorized nanocar inspired the design of nanosubmarines. We used fluorescence quenching and fluorescence correlation spectroscopy (FCS) to study the diffusion of single molecules. The fluorescence quenching experiments of Ru(bpy)3+2 by a quenching nanosubmarine was conducted, but no motor induced acceleration of the molecule were observed. Another fluorescent nanosubmarine was monitored by FCS, and no increase of diffusion coefficient was found. Finally, a 1-D channel approach was adopted for decreasing the effects of Brownian motion, and acceleration of nanosubmarine was observed.

Le trafic membranaire est un processus universel qui est essentiel pour la fonction neuronale dans un large spectre de fonctions. De la croissance neuronale et le développement morphologique à la libération des neurotransmetteurs et la plasticité synaptique, il prend en charge l'activité neuronale et donne d'innombrables questions qui animent la recherche sur la neurobiologie d'aujourd'hui. Notamment, l’exocytose des endosomes de recyclage (ER) dans les compartiments somatodendritiques participe à la transmission synaptique et à la potentialisation synaptique à long terme (PLT). Cependant la machine moléculaire sous-tendant l’exocytose des ER reste encore méconnue. Afin d’identifier les protéines SNAREs vésiculaires (v-SNARE) impliquées dans les différentes formes d’exocytose des ER postsynaptiques, nous avons d'abord imagé les protéines VAMP neuronales fusionnées avec la pHluorine, une GFP mutée sensible au pH dans les neurones de l’hippocampe en culture. Nous avons constaté que seulement VAMP2 et VAMP4, mais pas VAMP7, rapportaient des événements d’exocytose somatodendritique dans les neurones matures. Après avoir identifié ces deux protéines candidates, nous avons utilisé la combinaison de différentes techniques de régulation négative chronique ou aiguë pour désactiver leur fonction et observer les conséquences sur l’exocytose des ER, la transmission synaptique basale ou la PLT. Nos résultats suggèrent que VAMP2 est impliqué dans une forme d’exocytose régulée importante pour la PLT, mais pas l’exocytose constitutive des récepteurs AMPA, qui stabilise la transmission basale. VAMP4 est nécessaire pour l'exocytose constitutive d'une grande partie des endosomes, mais l'implication fonctionnelle de ces endosomes doit encore être explorée, car la régulation négative de VAMP4 ne modifie pas la transmission basale. / Membrane trafficking is a universal process that is essential for neuronal function in a wide spectrum of applications. From neuronal growth and morphological development to neurotransmitter release and synaptic plasticity, it supports neuronal activity and gives countless questions that drive today’s neurobiology research. Notably, the trafficking of recycling endosomes (REs) in somatodendritic compartments participates in synaptic transmission and plasticity, such as long-term synaptic potentiation (LTP). However, the fusion machinery mediating RE exocytosis is still unclear. To identify the vesicular SNAREs (v-SNAREs) involved in different forms of postsynaptic RE exocytosis, we first imaged neuronal VAMP proteins fused with pH-sensitive pHluorin in cultured hippocampal neurons, and found that only VAMP2 and VAMP4, but not VAMP7, underwent somatodendritic exocytosis in mature neurons. After identifying these two candidate proteins, we used a combination of different downregulation techniques to chronically or acutely deactivate their function and observe consequences on REs exocytosis, basal synaptic transmission and LTP. Our results suggest that VAMP2 is involved in activity-regulated exocytosis important for LTP, but not constitutive postsynaptic AMPARs exocytosis, supporting basal transmission. VAMP4 is required for constitutive exocytosis of at least a large proportion of REs, but the functional implication of these endosomes still need to be explored, as VAMP4 downregulation did not alter basal synaptic transmission.

The diploma thesis is focused on the design and realization of an experimental device. The purpose of device is the simulation of dynamic and kinematic conditions of knee replacement during the walking cycle. The simulator will be used to study the formation of the lubricating film in contact of the femoral component and the polyethylene spacer, and to the cyclic wear tests for knee replacements. First part of thesis is focused on the description of working parameters of knee replacements and analysis of knee joint simulators. The description of conceptual design and selection of the final variation follows. The final design allows simulation of dynamic and kinematic conditions of ISO 14 243-3. Creation of lubricating film is analyzed by the non-contact optical fluorescence microscopy method. The work includes complete drawing documentation, wiring diagram, verification of the functionality of the device and detailed operating instructions.

Given diploma thesis deals with the design and realization of a device whose purpose is to simulate real condition in the contact area of deep groove ball bearings. Direct observation of such area was then carried out. Initial part of the thesis addresses principles occurring in ball bearings, such as friction and lubrication and wear mechanisms. Several devices which are commonly used for bearing lubrication studies are then described at the work. Subsequently, author’s solution was introduced with the output of Ball-on-ring device. Ball-on-ring configuration is key feature due to its geometry since it enables to create identical conditions as those which occur in ball bearings such as contact pressure and velocities. Lubricant distribution by the contact area is analysed with fluorescent microscopy. Part of this thesis is a validation experiment and drawing documentation of the device.

In cancer research, there is a need to make accurate spatial measurements in multi-layered fluorescence microscopy images. Researchers would like to measure distances in and between biological objects such as nerves and tumours, to investigate questions which includes if nerve distribution in and around tumours can have a prognostic value in cancer diagnostics. This thesis is split into two parts, the first being: given arbitrary florescent images of cancer tissue samples, investigate the feasibility of automatically identifying nerves, tumours and blood vessels using classic image analysis. The second part is: given an image with identified objects, quantify their spatial data. By analysing 58 different cancer tissue samples we found that a modified Otsu method gives the most promising results for image segmentation. We found that non-verifiable objects and verifiable objects share the same pixel intensity distributions which implies that it is in general not possible to solely use thresholding methods to separate them from each other. For the spatial analysis, two measurement methods were introduced. An object based method that provides measurements from the edges of nerves to tumour edges, and a pixel based measurement method, which provides fraction based measurements that are comparable between different tissue samples.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

Dizertační práce se zabývá mechanismy mazání v náhradách kyčelního kloubu. Byla provedena systematická studie formování proteinového filmu při zahrnutí různých materiálů a provozních podmínek. Hlavní pozornost je přitom věnována roli jednotlivých proteinů obsažených v synoviální kapalině při současné přítomnosti dalších proteinů. Jelikož metody aplikované v předchozích studiích neumožňovaly separovat jednotlivé složky maziva, byla vyvinuta optická měřící metoda na principu fluorescenční mikroskopie. Z důvodu verifikace metody byly provedeny dvě nezávislé studie zaměřené na měření tloušťky mazacího filmu a dělení maziva na výstupu mazaného kontaktu. Z důvodu určitých limitací fluorescenční mikroskopie byla dále využita i metoda optické interferometrie, jejíž využití je ilustrováno ve studii zabývající se formováním mazacího filmu v náhradách kyčelního klubu při uvažování reálné konformity třecích povrchů. Závěrečná část práce představuje nový metodologický přístup založený na in situ pozorování kontaktní oblasti umožňující popsat roli jednotlivých proteinů ve vztahu k vývoji tloušťky mazacího filmu. Práce obsahuje originální výsledky, které přináší nové poznání v oblasti biotribologie náhrad kyčelního kloubu vedoucí k dalšímu vývoji implantátů při snaze zabránit jejich selhání v důsledku omezené životnosti.

Adenosine Triphosphate (ATP) can be released as a signal between cells in an autocrine and paracrine manner that binds purinergic receptors. Highly conserved, purinergic receptors expressed on the cell surface of neurons and astrocytes are capable of being activated across eight orders of magnitude from hundreds of nanomolar ATP to millimolar. Genetically encoded fluorescent protein biosensors have been used to detect ATP outside the cell, but a high affinity extracellular ATP sensor is required to study the ATP signaling dynamics from nanomolar to micromolar magnitudes. Previously, our lab developed a first generation sensor of extracellular ATP called ECATS1 (Conley et al.). To develop an improved sensor, we caried out site-directed mutagenesis of the sensor's ATP binding site and identified a mutant that exhibited a 4-fold increase in ATP binding affinity in solution. We then optimized the membrane-tethering of the sensor to achieve the 4-fold increase in extracellular ATP binding affinity when measured on live cell.s This second-generation sensor was dubbed ECATS2. As a proof-of-concept application, we sought to detect ATP release from cells using <i>in vitro</i> models of edema. We subjected HEK293A cells to hypo-osmotic shock (HOS), revealing ATP release at micromolar levels. Then we tested HOS in cultured cortical astrocytes, also revealing micromolar ATP release. However, when we tested neuron-astrocyte co-cultures, we no longer observed ATP release in response to HOS. Interestingly, this implies that co-culture either entirely prevented ATP release from astrocytes or dampened it into the nanomolar range below the limit of ECATS2 detection. Thus, we have validated the development of a higher affinity, second-generation sensor and used it to discover that ATP release from astrocytes after HOS can be affected by the presence of neurons. <br>