Psr1p interacts with SUN/sad1p and EB1/mal3p to establish the bipolar spindle

Regular Abstracts - Sunday Poster Presentations: no. 382During mitosis, interpolar microtubules from two spindle pole bodies (SPBs) interdigitate to create an antiparallel microtubule array for accommodating numerous regulatory proteins. Among these proteins, the kinesin-5 cut7p/Eg5 is the key player responsible for sliding apart antiparallel microtubules and thus helps in establishing the bipolar spindle. At the onset of mitosis, two SPBs are adjacent to one another with most microtubules running nearly parallel toward the nuclear envelope, creating an unfavorable microtubule configuration for the kinesin-5 kinesins. Therefore, how the cell organizes the antiparallel microtubule array in the first place at mitotic onset remains enigmatic. Here, we show that a novel protein psrp1p localizes to the SPB and plays a key role in organizing the antiparallel microtubule array. The absence of psr1+ leads to a transient monopolar spindle and massive chromosome loss. Further functional characterization demonstrates that psr1p is recruited to the SPB through interaction with the conserved SUN protein sad1p and that psr1p physically interacts with the conserved microtubule plus tip protein mal3p/EB1. These results suggest a model that psr1p serves as a linking protein between sad1p/SUN and mal3p/EB1 to allow microtubule plus ends to be coupled to the SPBs for organization of an antiparallel microtubule array. Thus, we conclude that psr1p is involved in organizing the antiparallel microtubule array in the first place at mitosis onset by interaction with SUN/sad1p and EB1/mal3p, thereby establishing the bipolar spindle.postprin

Removal of antagonistic spindle forces can rescue metaphase spindle length and reduce chromosome segregation defects

Regular Abstracts - Tuesday Poster Presentations: no. 1925Metaphase describes a phase of mitosis where chromosomes are attached and oriented on the bipolar spindle for subsequent segregation at anaphase. In diverse cell types, the metaphase spindle is maintained at a relatively constant length. Metaphase spindle length is proposed to be regulated by a balance of pushing and pulling forces generated by distinct sets of spindle microtubules and their interactions with motors and microtubule-associated proteins (MAPs). Spindle length appears important for chromosome segregation fidelity, as cells with shorter or longer than normal metaphase spindles, generated through deletion or inhibition of individual mitotic motors or MAPs, showed chromosome segregation defects. To test the force balance model of spindle length control and its effect on chromosome segregation, we applied fast microfluidic temperature-control with live-cell imaging to monitor the effect of switching off different combinations of antagonistic forces in the fission yeast metaphase spindle. We show that spindle midzone proteins kinesin-5 cut7p and microtubule bundler ase1p contribute to outward pushing forces, and spindle kinetochore proteins kinesin-8 klp5/6p and dam1p contribute to inward pulling forces. Removing these proteins individually led to aberrant metaphase spindle length and chromosome segregation defects. Removing these proteins in antagonistic combination rescued the defective spindle length and, in some combinations, also partially rescued chromosome segregation defects. Our results stress the importance of proper chromosome-to-microtubule attachment over spindle length regulation for proper chromosome segregation.postprin

Immuno Magnetic Thermosensitive Liposomes For Cancer Therapy

The present work describes the encapsulation of the drug doxorubicin (DOX) in immuno paramagnetic thermosensitive liposomes. DOX is the most common chemotherapeutic agent for the treatment of a variety of carcinomas. However, the pure drug has high cytotoxicity and therefore requires a targeted and biocompatible delivery system. The introduction includes concepts, modalities, and functionalities of the project. First, a detailed description of the cell type (triple-negative breast cancer) is given. Furthermore, the importance of liposomal doxorubicin is explained and the current state of research is shown. The importance of modification to achieve thermosensitive properties and the procedure for co-encapsulation with Gd chelate to achieve paramagnetic properties is also discussed. In addition, the first part describes the surface modification with ADAM8 antibodies, which leads to improved targeting. The second part of the thesis covers the different materials and methods used in this paper. The production of the liposomes LipTS, LipTS-GD, LipTS-GD-CY, LipTS-GD-CY-MAB and the loading of DOX using an ammonium sulfate gradient method were described in detail. The results part deals with the physicochemical characterization using dynamic light scattering and laser Doppler velocimetry, which confirmed a uniform monodisperse distribution of the liposomes. These properties facilitate the approach of liposomes to target cancer cells. The influence of lipid composition of liposomes, co-encapsulation with Gd chelate and surface modification of liposomes was evaluated and described accordingly. The size and structure of the individual liposomal formulations were determined by atomic force microscopy and transmission electron microscopy. Morphological examination of the liposomes confirmed agreement with the sizes obtained by dynamic light scattering. Temperature-dependent AFM images showed an intact liposome structure at 37 °C, whereas heating by UHF-MRI led to a lipid film indicating the destruction of the lipid bilayer. Furthermore, TEM images showed the morphological properties of the liposomes and gave a more precise indication of how Gd-chelate accumulates within the liposomes. Liposomes with Gd-chelate showed well-separated vesicles, suggesting that Gd- chelate is deposited in the lipid bilayer of the liposomes. Gd was encapsulated in the hydrophilic core whereas chelate was extended into the lipid bilayer. By differential scanning calorimetry and drug release, the heat-sensitive functionality of the liposomes could be determined. Liposomes showed a beginning of phase transition temperature at about 38 °C, which can be achieved by UHF-MRI exposure. The maximum phase transition temperature in the case of LipTS-GD and LipTS-GD-CY-MAB was 42 °C and 40 °C, respectively. A proof of concept study for the thermosensitive properties of liposomes and a time-dependent DOX release profile in hyperthermia was performed. Gd-chelate is encapsulated in both LipTS-GD and LipTS-GD-CY-MAB and led to paramagnetic properties of the liposomes. This facilitates imaging mediated DOX delivery and diagnosis of the solid tumor and metastatic cells. The change in relaxation rate R1 of liposomes was quantified before and after heating above Tm (T> Tm). The relaxivity of the liposomes was obtained from the adapted slope of the relaxation rate against the Gd concentration. Remarkably, the relaxation rate and relaxivity increased after heating the liposomes above Tm (T> Tm), suggesting that the liposomes opened, released Gd chelate, and the exchange of water molecules became faster and more practicable. Toxicity studies describe the different mechanisms for induced DOX toxicity. The increased cytotoxic effect at elevated temperatures showed that the induced toxicity is thermally dependent, i.e. DOX was released from the liposomes. The high viability of the cells at 37 °C indicates that the liposomes were intact at normal physiological temperatures. Under UHF-MRI treatment, cell toxicity due to elevated temperature was observed. The cellular uptake of liposomes under UHF-MRI was followed by a confocal laser scanning microscope. An increase in fluorescence intensity was observed after UHF-MRI exposure. The study of the uptake pathway showed that the majority of liposomes were mainly uptake by clathrin-mediated endocytosis. In addition, the liposomes were modified with anti-ADAM8 antibodies (MAB 1031) to allow targeted delivery. The cellular binding capabilities of surface-modified and non-modified liposomes were tested on cells that had ADAM8 overexpression and on ADAM8 knockdown cells. Surface-modified liposomes showed a significant increase in binding ability, indicating significant targeting against cells that overexpress ADAM8 on their surface. In addition, cells with knockdown ADAM8 could not bind a significant amount of modified liposomes. The biocompatibility of liposomes was assessed using a hemolysis test, which showed neglected hemolytic potential and an activated thromboplastin time (aPTT), where liposomes showed minimal interference with blood clotting. Hemocompatibility studies may help to understand the correlation between in vitro and in vivo. The chorioallantois model was used in ovo to evaluate systematic biocompatibility in an alternative animal model. In the toxicity test, liposomes were injected intravenously into the chicken embryo. The liposomes showed a neglectable harmful effect on embryo survival. While free DOX has a detrimental effect on the survival of chicken embryos, this confirms the safety profile of liposomes compared to free DOX. LipTS-GD-CY-MAB were injected into the vascular system of the chicken embryo on egg development day 11 and scanned under UHF-MRI to evaluate the magnetic properties of the liposomes in a biological system with T2-weighted images (3D). The liposomal formulation had distinct magnetic properties under UHF MRI and the chick survived the scan. In summary, immunomagnetic heat-sensitive liposomes are a novel drug for the treatment of TNBC. It is used both for the diagnosis and therapy of solid and metastasizing tumors without side effects on the neighboring tissue. Furthermore, a tumor in the CAM model will be established. Thereafter, the selective targeting of the liposomes will be visualized and quantitated using fluorescence and UHF-MRI. Liposomes are yet to be tested on mice as a xenograft triple-negative breast cancer model, in which further investigation on the effect of DOX-LipTS-GD-CY-MAB is evaluated. On one hand, the liposomes will be evaluated regarding their targetability and their selective binding. On the other hand, the triggered release of DOX from the liposomes after UHF-MRI exposure will be quantitated, as well as evaluate the DOX-Liposomes therapeutic effect on the tumor

Sertoli cells have a functional NALP3 inflammasome that can modulate autophagy and cytokine production

Sertoli cells, can function as non-professional tolerogenic antigen-presenting cells, and sustain the blood-testis barrier formed by their tight junctions. The NOD-like receptor family members and the NALP3 inflammasome play a key role in pro-inflammatory innate immunity signalling pathways. Limited data exist on NOD1 and NOD2 expression in human and mouse Sertoli cells. Currently, there is no data on inflammasome expression or function in Sertoli cells. We found that in primary pre-pubertal Sertoli cells and in adult Sertoli line, TLR4\NOD1 and NOD2 crosstalk converged in NF?B activation and elicited a NALP3 activation, leading to de novo synthesis and inflammasome priming. This led to caspase-1 activation and IL-1? secretion. We demonstrated this process was controlled by mechanisms linked to autophagy. NOD1 promoted pro-IL-1? restriction and autophagosome maturation arrest, while NOD2 promoted caspase-1 activation, IL-1? secretion and autophagy maturation. NALP3 modulated NOD1 and pro-IL-1? expression, while NOD2 inversely promoted IL-1?. This study is proof of concept that Sertoli cells, upon specific stimulation, could participate in male infertility pathogenesis via inflammatory cytokine induction

Das geprägte Kcnk9-Gen der Maus - Expressionsanalyse in spezifischen Hirnregionen und phänotypische Analyse von Knockout-Mäusen

VIII, 158 Seite

SPATIAL ORGANIZATION OF LAMIN A/C IN HODGKIN’S LYMPHOMA AND MULTIPLE MYELOMA

Genomic Instability is a complex phenomenon observed both at the DNA and the chromosome level. It is responsible for the generation of mutations and chromosomal rearrangements, which can potentially lead to tumor development. A common genomic instability feature found in some malignant tumors is the shortening of the telomeres, the end of the chromosomes. Shortening of telomeres leads to the formation of anaphase bridges, subsequent breakage and initiation new breakage-bridge-fusion (BFB) cycles. Classical Hodgkin’s lymphoma (cHL) and Multiple Myeloma (MM) are two blood tumor both characterized by genomic instability. cHL is a B-Cell lymphoma comprised of mononuclear Hodgkin cells (H) and bi- to multi-nucleated Reed-Sternberg (RS) cells, both characterized by numerical (nCIN) and structural chromosomal instability (sCIN). Transition from H to RS cells is correlated with aberrant number of mitotic spindles, aberrant 3D telomere organization, increase in the number of telomere aggregates and telomere loss. Advanced shortening of telomeres leads disruption of 3D interaction of the telomere with TRF2, a member of the shelterin proteins responsible for the protection of the chromosome ends from the DNA damage repair system. MM, an incurable plasma cell disorder, is also characterized by CIN, microsatellite instability (MSI), and telomere attrition. Lamin A/C, a nuclear envelope protein and component of the nuclear matrix, is important for the assembly of the mitotic spindles and for the maintenance of the 3D telomeres architecture by binding the shelterin protein TRF2. Due to lamin A/C involvement in the maintenance of genomic stability, our goal was to investigate lamin A/C 3D spatial organization in cHL and MM, and understand whether the localized uncapping of TRF2 from telomeres leads to their detachment from the nuclear matrix component lamin A/C causing genomic instability. Western blot analysis and qRT-PCR analysis revealed that H and RS are characterized by an overall overexpression of lamin A/C when compared to the B-lymphocytes control. Three dimensional (3D) fluorescent microscopy in cHL and MM cells revealed that the regular homogeneous lamin A/C pattern identified in normal activated B-lymphocytes, was replaced by an aberrant lamin A/C 3D spatial distribution characterized by the presence of internal lamin A/C structures. Different lamin A/C patterns where observed where the internal lamin A/C sub-divided the nucleus in to two or multiple compartments, suggesting a possible involvement of lamin A/C in the multi-nucleation process. To investigate lamin A/C involvement in the multinucleation process and transition from H to RS, the downregulation of lamin A/C with siRNA was performed. Downregulation of lamin A/C affected the transition from H to RS cells, as the number of RS cells found after lamin silencing was decreased. Telomere 3D structure and nuclear organization were also affected by the deregulation of lamin A/C. H and RS cells were also characterized by unbinding of telomere-TRF2-lamin A/C when compared to the healthy control. Lack of co-localization of telomere-TRF2-lamin A/C confirms lamin A/C involvement in genomic instability, and suggests that localized absence of TRF2 could be causal to the genome remodeling in cHL

Optical probing of spatial structural abnormalities in cells/tissues due to cancer, drug-effect, and brain abnormalities using mesoscopic physics-based spectroscopic techniques

The quantitative measurement of structural alterations at the nanoscale level is important for understanding the physical states of weakly disordered optical mediums such as cells/tissues. Progress in certain diseases, such as cancer or abnormalities in the brain, is associated with the nanoscale structural alterations at basic building blocks of the cells/tissues. Elastic light scattering, especially at visible wavelengths range provides non-invasive ways to probe the cells/tissues up to nanoscale level. Therefore, a mesoscopic physics-based open light scattering technique with added finer focusing, partial wave spectroscopy (PWS), is developed to probe nanoscale changes. Then, molecular-specific light localization technique, a close scattering approach called inverse participation ratio (IPR) is proposed that is sensitive to nano to microstructural cell/tissue alterations. In this dissertation, we have introduced the further engineered PWS system with the finer focus for precise volume scattering and molecular-specific light localization IPR techniques. As an application of PWS, we first probe precise scattering volume in commercially available tissue microarrays (TMA) tissue samples to standardize the existing cancer diagnostic methods by distinguishing the cancer stages. We also apply the PWS technique to probe chemotherapy drug-treated metastasizing cancer patients by xenografting prostate cancer cells using a mouse model and identify drug-sensitive and drug-resistance treatment cases. On the other hand, as an illustration of another mesoscopic physics-based molecular specific light localization technique, Confocal-IPR, we study the effects of a probiotic on chronic alcoholic mice brains by targeting the molecular specific alteration in glial cells, astrocytes and microglia, and chromatin of the brain cells through staining with appropriate dyes/proteins. Using structural disorder of IPR as a biomarker, the results show that probiotics in the presence of alcohol are beneficial and help overall brain health. Finally, a TEM-IPR study was performed using nanoscale resolution TEM imaging to support the optical IPR method by studying the anti-cancerous drug effect in ovarian cancer cells. The result shows that we can quantitatively measure the effect of anti-cancerous drugs in cancer treatment and the level of tumorigenicity far below the diffraction limit, and it has a similar effect and supports the optical IPR method

Diffusion-weighted Imaging of Lymph Node Tissue

Purpose: The study investigates the hypothesis of clinically observed decreased apparent diffusion coefficient (ADC) of cancerous lymph nodes can be attributed to increased cellularity. The study characterises the mean diffusivity (MD) of lymph node sub-structures and investigates correlation between MD and cellularity metrics. The study also investigates the theoretical information content of single and multi-biophysical models. Methods:. A 3 mm diameter core sample was extracted from a formalin fixed lymph node tissue post-surgery and imaged using 9.4T and 16.4T Bruker MRI system. Samples were sectioned and stained with haematoxylin and eosin (H&E). Diffusion tensor model was fitted voxelwise and MD values were computed using Matlab. Cellularity metrics includes measurement of nuclear count and nuclear area. Eleven models with combinations of isotropic, anisotropic, and restricted components were tested for diffusion modelling and ranked using the Akaike information criterion (AIC). Results: The findings showed distinct diffusivities of lymph node sub-structures (capsule and parenchyma). Parenchyma in normal lymph node tissues had higher MD (0.71 ± 0.17 µm2/ms) than metastatic parenchyma (0.52 ± 0.08 µm2/ms) and lymphoma (0.47 ± 0.19 µm2/ms). No correlation were observed between MD and nuclear count (r = 0.368) and nuclear area (r = 0.368) respectively at 95 % confidence intervals. The single biophysical models (ADC and DTI) were ranked lowest by AIC. Multi-biophysical models consist of anisotropic and restricted diffusion (Zeppelin-sphere, Ball-stick-sphere, and Ball-sphere) were ranked highest in the majority of voxels of the tissue samples. Conclusion: A distinct diffusivity value were found in lymph node sub-structures with no correlation to cellularity. Multi-biophysical models were ranked highest and extract more information from the measurement data than simple single biophysical models

Characterization of Metastasis-Associated Cell Surface Glycoproteins in Prostate Cancer

Prostate cancer (PCa) is a major health problem in males in the United States. Its lethality is mostly attributed to the primary tumor metastasizing to distant sites that are highly resistant to conventional therapies. Serum Prostate Specific Antigen (PSA) is the only protein biomarker used in clinic for prediction of prostate cancer recurrence following local therapies. Nonetheless, PSA lacks the ability to predict the behavior of an individual tumor in an individual patient. Therefore, development of reliable biomarkers for detection of metastatic potential in primary tumors, as well as discovery of new therapeutic targets, is in a great need for improved disease survival and management. Tumor metastasis is a multistep process involving extravasation of a cancer cell subsequent invasion and expression at a site distal to the primary tumors. Cell surface glycoproteins play pivotal roles as recognition molecules in a range of cell communication and adhesion events. Aberrant cell surface glycosylation has been reported in various cancers including PCa, and strongly correlated with prognosis and metastasis. However, the staggering complexity of glycans renders their analysis extraordinarily difficult. This research project aims to develop a mass spectrometry-based glycoproteomic approach for the selective isolation and identification of cell surface glycoproteins from cellular samples, and apply this technology to the discovery of new glycoprotein biomarkers which are indicative of prostate cancer progression and metastasis. To this end, cell surface glycosylation patterns were characterized by lectin flow cytometry and lectin cytochemistry on a human syngeneic PCa cell metastatic model, PC3 and its two variants with different metastatic potentials. It was found that metastatic potentials of PC3 variants were inversely correlated with cell surface α2-6 sialic acid levels. Targeted to cell surface sialoglycoproteins, a new glycoproteomic approach was successfully developed, which combined selective metabolic labeling of cell surface sialyl glycans, chemically probing the labeled sugar with a biotin tag, affinity purification of sialylated proteins, SDS-PAGE separation, and subsequent LC-MS/MS for protein identification. Application of this methodology in our prostate cancer model system resulted in unique identification of a total of 80 putative cell surface sialoglycoproteins differentially expressed between PC3 variants. After prioritization of the candidate biomarkers, one cell-based prioritized biomarker CUB-domain-containing protein 1 (CDCP1) was verified in prostate cancer cell lines and clinical samples, including tissues and body fluids, by immunoassays. Results indicated that expression of CDCP1 protein is dysregulated in prostate cancer and it has potential utility as a therapeutic target and a diagnostic marker for PCa progression. Overall, the data from this research project provided the proof-of-principle evidence for our targeted glycoproteomic approach, which we believe will help expedite the discovery of new cancer biomarkers and therapeutic targets in diseases and delineation of signal transduction pathways on a global scale

Effects of shear stress on NF-κB transcription factors in vascular endothelium

Atherosclerosis, a chronic inflammatory disease of arteries, occurs predominantly at regions of the arterial system that are exposed to disturbed patterns of blood flow. Blood flow influences the atherosclerosis by exerting shear stress on endothelial cells (ECs). Although the signalling pathways that activate pro-inflammatory NF-κB transcription factors are well defined, the regulation and physiological significance of differential NF-κB subunit expression is poorly understood. In this thesis, we demonstrate that RelA NF-κB sub-unit expression is positively regulated in ECs via c-Jun N-terminal kinase (JNK) and the transcription factor ATF2. This pathway promoted focal arterial inflammation as genetic deletion of JNK1 reduced RelA expression and macrophage accumulation at an athero-susceptible site. Furthermore, JNK signalling to RelA is controlled by mechanical forces as en face immunostaining revealed that disturbed flow patterns (generated by a constrictive cuff) elevated RelA expression in murine carotid arteries via JNK1. Positron emission tomography and en face staining revealed that disturbed flow enhanced 18F-fluorodeoxyglucose uptake (a marker of inflammation) and accumulation of CD68-positive inflammatory cells in arteries via JNK1. We conclude that disturbed flow promotes arterial inflammation via a novel JNK-NF-κB cross-talk. The duration of RelA nuclear localisation is an important determinant of the magnitude and specificity of target gene expression. En face staining revealed that RelA rapidly accumulated in the nucleus upon LPS stimulation in ECs at both athero-protected and athero-susceptible sites. RelA was exported from the nucleus to the cytoplasm in response to prolonged stimulation in the athero-protected region but not in the athero-susceptible region. The duration of RelA nuclear localisation was suppressed by histone deacetylases which displayed higher activity at the protected site compared to the susceptible site. Overall, our findings reveal that ECs at athero-susceptible sites are primed for inflammatory activation via complementary mechanisms that enhance both the expression and the activity of NF-κB transcription factors