Studies on the adhesion properties of the liver-metastatic murine RAW117 large-cell lymphoma cells: Roles of $\beta\sb3$ integrin

Metastasis is the major cause of death in cancer patients. Since many cancers show organ-preference of metastasis, elucidation of the underlying mechanisms of metastasis will benefit diagnosis or treatment of metastatic diseases. Adhesion mechanisms are thought to be involved in organ-preference of metastasis, because metastatic cells show organ preference in adhering to organ-derived microvascular endothelial cells. The adhesion molecules in this process remain largely unidentified. I have examined a series of murine RAW117 large-cell lymphoma cells variants selected in vivo for liver-colonizing properties ($\rm{H10{>>}L17>P}$). The highly liver-metastatic H10 cells were found to differentially express much higher levels of integrin $\alpha\rm\sb{v}\beta\sb3$ than L17 or P cells. H10 cells also adhered at higher rates to vitronectin and fibronectin than to fibrinogen, fibrin, laminin and type I collagen, and adhered at significantly higher rates to (GRGDS)$\sb4$ than to monomeric RGD-peptides. In contrast, P and L17 cells did not adhere well to the above substrates. H10 cells also spread well on vitronectin and migrated toward vitronectin concentration gradients. Pretreament of H10 cells with anti-$\beta\sb3$ monoclonal antibodies resulted in significant decreases in adhesion of H10 cells to vitronectin and immobilized (GRGDS)$\sb4$, and reduced the formation of experimental liver metastases in syngeneic Balb/c mice.^ Adhesion of RAW117 cells under hydrodynamic shear stresses was also studied because tumor cell adhesion occurs under fluid shear stresses in target organ microvessels. Similar to their properties found with static adhesion assays, H10 cells stabilized their hydrodynamic adhesion to vitronectin, fibronectin and (GRGDS)$\sb4$ much more quickly than P or L17 cells. Unlike their static adhesion properties, RAW117 cells showed differential adhesion stabilization to liver-sinusoidal endothelial cell-derived extracellular matrix ($\rm{H10{>>}L17>P}$). Although not supporting static adhesion of RAW117 cells, monomeric RGD-peptides mediated adhesion stabilization of H10 cells but not L17 or P cells. Integrin $\rm\alpha\sb{v}\beta\sb3$ was found to be involved in stabilizing H10 cell adhesion to vitronectin, (GRGDS)$\sb4$, monomeric RGD-peptide R1, and liver sinusoidal endothelial cell-derived extracellular matrix.^ This study is the first to provide evidence that integrin $\rm\alpha\sb{v}\beta\sb3$ is differentially expressed in liver-metastatic lymphoma cells and involved in differential adhesion of these cells. The results indicate that strong static adhesion and especially the unique hydrodynamic adhesion of RAW117 cells to the RGD-containing substrates correlate with liver-metastatic potentials. Thus, integrin $\rm\alpha\sb{v}\beta\sb3$ may play an important role in liver-preferential metastasis of RAW117 large-cell lymphoma cells. ^

Molecular epidemiology of tuberculosis in Cambodian children

SUMMARY We analysed Mycobacterium tuberculosis strains from children, hospitalized from January 2004 to July 2008 in the largest paediatric hospital complex in Cambodia. Specimens were tested for drug susceptibility and genotypes. From the 260 children, 161 strains were available. The East African-Indian genotype family was the most common (59·0%), increasing in frequency with distance from the Phnom Penh area, while the frequency of the Beijing genotype family strains decreased. The drug resistance pattern showed a similar geographical gradient: lowest in the northwest (4·6%), intermediate in the central (17·1%), and highest in the southeastern (30·8%) parts of the country. Three children (1·9%) had multidrug-resistant tuberculosis. The Beijing genotype and streptomycin resistance were significantly associated (P < 0·001). As tuberculosis in children reflects recent transmission patterns in the community, multidrug resistance levels inform about the current quality of the tuberculosis programme.

Molecular epidemiology of hepatitis B virus infection in Switzerland: a retrospective cohort study.

BACKGROUND Chronic hepatitis B virus (HBV) infection affects up to 7 % of the European population. Specific HBV genotypes are associated with rapid progression to end-stage liver disease and sub-optimal interferon treatment responses. Although the geographic distribution of HBV genotypes differs between regions, it has not been studied in Switzerland, which lies at the crossroads of Europe. METHODS In a retrospective analysis of 465 HBV samples collected between 2002 and 2013, we evaluated the HBV genotype distribution and phylogenetic determinants, as well as the prevalence of serological evidence of hepatitis delta, hepatitis C and HIV infections in Switzerland. Baseline characteristics of patients were compared across their region of origin using Fisher's exact test and ANOVA, and risk factors for HBeAg positivity were assessed using logistic regression. RESULTS The Swiss native population represented 15.7 % of HBV-infected patients living in Switzerland. In the overall population, genotype D was most prevalent (58.3 %), whereas genotype A (58.9 %) was the predominant genotype among the Swiss native population. The prevalence of patients with anti-HDV antibodies was 4.4 %. Patients of Swiss origin were most likely to be HBeAg-positive (38.1 %). HBV genotypes of patients living in Switzerland but sharing the same original region of origin were consistent with their place of birth. CONCLUSIONS The molecular epidemiology of HBV infection in Switzerland is driven by migration patterns and not by the genotype distribution of the native population. The prevalence of positive anti-HDV antibodies in our cohort was very low.

Molecular mechanism of PUVA-induced apoptosis in mouse epidermal cells

A combination of psoralens and ultraviolet-A radiation referred to as PUVA, is widely used in the treatment of psoriasis. PUVA therapy is highly effective in killing hyperproliferative cells, but its mechanism of action has not been fully elucidated. Psoralen binds to DNA, and upon photoactivation by UVA, it forms monofunctional adducts and interstrand cross-links. PUVA treatment has been shown to be mutagenic and to produce tumors in animals. In addition, epidemiological studies have reported a 10 to 15 percent increased risk of developing squamous cell carcinoma in individuals treated chronically with PUVA. However, it remains a treatment for skin disorders such as psoriasis because its benefits outweigh its risks. The widespread use of PUVA therapy and its associated cancer risk requires us to understand the molecular mechanisms by which PUVA induces cell death. Immortalized JB6 mouse epidermal cells, p53−/− mice, and Fas Ligand−/− (gld) mice were used to investigate the molecular mechanism by which PUVA kills cells. Treatment of JB6 cells with 10 μg/ml 8-methoxypsoralen followed by irradiation with 20 kJ/m2 UVA resulted in cell death. The cells exhibited morphological and biochemical characteristics of apoptosis such as chromatin condensation, DNA ladder formation, and TUNEL-positivity. PUVA treatment stabilized and phosphorylated p53 leading to its activation, as measured by nuclear localization and induction of p21Waf/Cip1, a transcriptional target of p53. Subsequent in vivo studies revealed that there was statistically significantly less apoptosis in p53 −/− mice than in p53+/+ mice at 72 hours after PUVA. In addition, immunohistochemical analysis revealed more Fas and FasL expression in p53+/+ mice than in p53−/− mice, suggesting that p53 is required to transcriptionally activate Fas, which in turn causes the cells to undergo apoptosis. Studies with gld mice confirmed a role for Fas/FasL interactions in PUVA-induced apoptosis. There was statistically significantly less apoptosis in gld mice compared with wild-type mice 24, 48, and 72 hours after PUVA. These results demonstrate that PUVA-induced apoptosis in mouse epidermal cells requires p53 and Fas/FasL interactions. These findings may be important for designing effective treatments for diseases such as psoriasis without increasing the patient's risk for skin cancer. ^

Signal relay between two integral membrane proteins: The sensory rhodopsin I/HtrI transducer molecular complex

The molecular complex of sensory rhodopsin I (SRI) and its transducer HtrI mediate color-sensitive phototaxis in the archaeon Halobacterium salinarum. Orange light causes an attractant response by a one-photon reaction and white light causes a repellent response by a two-photon reaction. Three aspects of this molecular complex were explored: (i) We determined the stoichiometry of SRI and HtrI to be 2:2 by gene fusion analysis. A SRI-HtrI fusion protein was expressed in H. salinarum and shown to mediate 1-photon and 2-photon phototaxis responses comparable to wild-type complex. Disulfide crosslinking demonstrated that the fusion protein is a homodimer in the membrane. Measurement of photochemical reaction kinetics and pH titration of absorption spectra established that both SRI domains are complexed to HtrI in the fusion protein, and therefore the stoichiometry is 2:2. (ii) Cytoplasmic channel closure of SRI by HtrI, an important aspect of their interaction, was investigated by incremental HtrI truncation. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. The closure activity is localized to 5 specific residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp76 counterion to the protonated Schiff base chromophore. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. (iii) We developed a procedure for reconstituting HtrI-free SRI and the SRI/HtrI complex into liposomes, which exhibit photocycles with opened and closed cytoplasmic channels, respectively, as in the membrane. This opens the way for study of the light-induced conformational change and the interaction in vitro by fluorescence and spin-labeling. Single-cysteine mutations were introduced into helix F of SRI, labeled with a nitroxide spin probe and a fluorescence probe, reconstituted into proteoliposomes, and light-induced conformational changes detected in the complex. The probe signals can now be used as the readout of signaling to analyze mutants and the kinetics of signal relay. ^

Carcinoma of the lower uterine segment: A newly described association with Lynch Syndrome

Purpose. We performed a case-comparison study to describe the characteristics of LUS tumors and their association with risk factors for endometrial cancer. ^ Patients and Methods. From January 1996 through October 2007, 3,892 women were identified with a diagnosis of primary endometrial carcinoma or primary cervical adenocarcinoma. Pathology records from the 1,009 women who had a hysterectomy were reviewed. Subjects were included in the LUS group only if the tumor was clearly originating from the area between the lower corpus and upper cervix in the hysterectomy specimen. The LUS group was compared to all patients with endometrial corpus carcinoma who underwent hysterectomy at our institution in a 12-month period randomly selected from the study period. Risk factors for endometrial carcinoma such as body mass index (BMI) and Lynch Syndrome were assessed. Expression of estrogen receptor (ER), vimentin, carcinoembryonic antigen (CEA), p16, and human papilloma virus DNA (HPV DNA) was assessed; this panel is known to be effective in distinguishing adenocarcinomas of endometrial versus endocervical origin. Fisher's Exact, Chi-square, Mann-Whitney, and Student's t-tests were utilized for statistical analysis. ^ Results. Thirty-five of 1,009 women had endometrial carcinoma of the LUS (3.5%; 95% CI: 2–4%). Compared to patients with corpus tumors, LUS patients were younger (54.2 vs. 62.9 years, P = .001), had higher stage (P < .001), and more invasive tumors (P = .001). Preoperative diagnosis of the LUS tumors more frequently included the possibility of endocervical adenocarcinoma ( P < .001), leading to preoperative radiation therapy in 4 patients. Median BMI was similar in the LUS and corpus groups. Seventy-three percent of the available LUS tumors had a similar immunohistochemical expression pattern to conventional endometrioid adenocarcinoma. Because of the young median age for the LUS group, we performed immunohistochemistry for Lynch syndrome-associated DNA mismatch repair proteins MLH1, MSH2, MSH6, and PMS2. Microsatellite instability testing (MSI) and MLH1 promoter hypermethylation were performed when indicated. Thirty-six percent of the LUS tumors were MSI-high. Ten of thirty-five (29%) women with LUS tumors were either confirmed to have Lynch Syndrome or were strongly suspected to have Lynch Syndrome based on tissue-based molecular assays (95% CI, 16 to 45%). ^ Conclusions. Endometrial carcinoma arising in the LUS is a clinical and pathologic entity which can be diagnostically confused with cervical adenocarcinoma. In general, LUS tumors can be correctly identified as being endometrial carcinoma using the immunohistochemical panel noted above. The prevalence of Lynch Syndrome in patients with LUS tumors is much greater than that of the general endometrial cancer population (1.8%) or in endometrial cancer patients younger than 50 years of age (8–9%). Based on our results, the possibility of Lynch Syndrome should be considered in women with LUS tumors. ^

Spectroscopic and functional investigations of the AMPA subtype of ionotropic glutamate receptors

Ion channels play a crucial role in the functioning of different systems of the body because of their ability to bridge the cell membrane and allow ions to pass in and out of the cell. Ionotropic glutamate receptors are one class of these important proteins and have been shown to be critical in propagating synaptic transmission in the central nervous system and in other diverse functions throughout the body. Because of their wide-ranging effects, this family of receptors is an important target for structure-function investigations to understand their mechanism of action. ^ α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are one subtype of glutamate receptors and have been shown to be the primary receptors involved in rapid excitatory signaling in the central nervous system. Agonist binding to the extracellular ligand binding domain of these receptors causes various conformational changes that culminate in formation of the ion channel. Previous structural investigations have provided important information about their mechanism of action, including uncovering a relationship between the degree of cleft closure in the binding domain and activation of the receptor. However, what question remains unanswered is how specific interactions between the agonist and the protein interplay with cleft closure to mediate receptor activation. ^ To investigate this question, I applied a multiscale approach to investigate the effects of agonist binding on various levels. Vibrational spectroscopy was utilized to investigate molecular-level interactions in the binding pocket, and fluorescence resonance energy transfer (FRET) was employed to measure cleft closure in the isolated ligand binding domain. The results of these studies in the isolated binding domain were then correlated to activation of the full receptor. These investigations showed a relationship between the strength of the interaction at the α-amine group of the agonist and extent of receptor activation, where a stronger interaction correlated to a larger activation, which was upheld even when the extent of cleft closure did not correlate to activation. These results show that this interaction at the α-amine group is critical in mediating the allosteric mechanism of activation and provide a bit more insight into how agonist binding is coupled to channel gating in AMPA receptors. ^

Report on the practice based culminating experience as teaching assistant for "Pathology and Public Health" class taught at University of Texas School of Public Health, Houston

Objectives. The objectives of this report were to describe current best standards in online education, class competencies, class objectives, class activities and to compare the class competencies, objectives and activities undertaken with the current best practices in online teaching and to provide a list of recommendations based on the most efficacious practices. ^ Methods. Utilizing the key words- online teaching, national standards, quality, online courses, I: (1) conducted a search on Google to find the best standard for quality online courses; the search yielded National Standards for Quality Online Teaching as the gold standard in online course quality; (2) specified class objectives and competencies as well as major activities undertaken as a part of the class. Utilizing the Southern Regional Education Board evaluation checklist for online courses, I: (1) performed an analysis comparing the class activities, objectives, and competencies with the current best standards; (2) utilized the information obtained from the analysis and class experiences to develop recommendations for the most efficacious online teaching practices. ^ Results. The class met the criteria set by the Southern Regional Education Board for evaluating online classes completely in 75%, partially in 16% and did not meet the criteria in 9% cases. The majority of the parameters in which the class did not meet the standards (4 of 5) were due to technological reasons beyond the scope of the class instructor, teaching assistant and instructional design. ^ Discussion. Successful online teaching requires awareness of technology, good communication, methods, collaboration, reflection and flexibility. Creation of an online community, engaging online learners and utilizing different learning styles and assessment methods promote learning. My report proposes that online teaching should actively engage the students and teachers with multiple interactive strategies as evidenced from current best standards of online education and my “hands-on” work experience. ^ Conclusion. The report and the ideas presented are intended to create a foundation for efficacious practice on the online teaching platform. By following many of the efficacious online practices described in the report and adding from their own experiences, online instructors and teaching assistants can contribute to effective online learning. ^

Characterization of fecal contamination targeting 16S rRNA bacteroides molecular markers in the Santa Cruz River, Arizona

Understanding the origins, transport and fate of contamination is essential to effective management of water resources and public health. Individuals and organizations with management responsibilities need to understand the risks to ecosystems and to humans from contact with contamination. Managers also need to understand how key contaminants vary over time and space in order to design and prioritize mitigation strategies. Tumacacori National Historic Park (NHP) is responsible for management of its water resources for the benefit of the park and for the health of its visitors. The existence of microbial contaminants in the park poses risks that must be considered in park planning and operations. The water quality laboratory at the Maricopa Agricultural Center (in collaboration with stakeholder groups and individuals located in the ADEQ-targeted watersheds) identified biological changes in surface water quality in impaired reaches rivers to determine the sources of Escherichia coli (E. coli); bacteria utilizing innovative water quality microbial/bacterial source tracking methods. The end goal was to support targeted watershed groups and ADEQ towards E. coli reductions. In the field monitoring was conducted by the selected targeted watershed groups in conjunction with The University of Arizona Maricopa Agricultural Center Water Quality Laboratory. This consisted of collecting samples for Bacteroides testing from multiple locations on select impaired reaches, to determine contamination resulting from cattle, human recreation, and other contributions. Such testing was performed in conjunction with high flow and base flow conditions in order to accurately portray water quality conditions and variations. Microbial monitoring was conducted by The University of Arizona Water Quality Laboratory at the Maricopa Agricultural Center using genetic typing to differentiate among two categories of Bacteroides: human and all (total). Testing used microbial detection methodologies and molecular source tracking techniques.^

TAZ AS A REGULATOR OF MESENCHYMAL TRANSFORMATION AND CLINICAL AGGRESSIVENESS IN GLIOMAS

Glioblastoma multiforme (GBM) is an aggressive, high grade brain tumor. Microarray studies have shown a subset of GBMs with a mesenchymal gene signature. This subset is associated with poor clinical outcome and resistance to treatment. To establish the molecular drivers of this mesenchymal transition, we correlated transcription factor expression to the mesenchymal signature and identified transcriptional co-activator with PDZ-binding motif (TAZ) to be highly associated with the mesenchymal shift. High TAZ expression correlated with worse clinical outcome and higher grade. These data led to the hypothesis that TAZ is critical to the mesenchymal transition and aggressive clinical behavior seen in GBM. We investigated the expression of TAZ, its binding partner TEAD, and the mesenchymal marker FN1 in human gliomas. Western analyses demonstrated increased expression of TAZ, TEAD4, and FN1 in GBM relative to lower grade gliomas. We also identified CpG islands in the TAZ promoter that are methylated in most lower grade gliomas, but not in GBMs. TAZ-methylated glioma stem cell (GSC) lines treated with a demethylation agent showed an increase in mRNA and protein TAZ expression; therefore, methylation may be another novel way TAZ is regulated since TAZ is epigenetically silenced in tumors with a better clinical outcome. To further characterize the role of TAZ in gliomagenesis, we stably silenced or over-expressed TAZ in GSCs. Silencing of TAZ decreased invasion, self-renewal, mesenchymal protein expression, and tumor-initiating capacity. Over-expression of TAZ led to an increase in invasion, mesenchymal protein expression, mesenchymal differentiation, and tumor-initiating ability. These actions are dependent on TAZ interacting with TEAD since all these effects were abrogated with TAZ could not bind to TEAD. We also show that TAZ and TEAD directly bind to mesenchymal gene promoters. Thus, TAZ-TEAD interaction is critically important in the mesenchymal shift and in the aggressive clinical behavior of GBM. We identified TAZ as a regulator of the mesenchymal transition in gliomas. TAZ could be used as a biomarker to both estimate prognosis and stratify patients into clinically relevant subgroups. Since mesenchymal transition is correlated to tumor aggressiveness, strategies to target and inhibit TAZ-TEAD and the downstream gene targets may be warranted in alternative treatment.

The Role of the Arched Helicases in Exosome-Mediated Function

RNA processing and degradation are two important functions that control gene expression and promote RNA fidelity in the cell. A major ribonuclease complex, called the exosome, is involved in both of these processes. The exosome is composed of ten essential proteins with only one catalytically active subunit, called Rrp44. While the same ten essential subunits make up both the nuclear and cytoplasmic exosome, there are nuclear and cytoplasmic exosome cofactors that promote specific exosome functions in each of the cell compartments. To date, it is unclear how the exosome distinguishes between RNA substrates. We hypothesize that compartment specific cofactors may promote the substrate specificity of the exosome. In this work, I characterize several cofactors of the exosome, both nuclear and cytoplasmic. First, I describe the arch domain, which is a unique domain in a nuclear and a cytoplasmic cofactor of the exosome. Specifically, I show that the arch domain of the nuclear exosome cofactor, Mtr4, is required for specific exosome-mediated activities and overlaps functionally with the exosome-associated exonuclease, Rrp6. Further, I show that the arch domain of Ski2 is required for the degradation of normal and aberrant mRNAs. Additionally, this work describes in detail the Mtr4 domains involved in the physical association with other RNA processing proteins. Further, I characterize the minimal Mtr4-binding region in a third exosome cofactor, Trf5. Understanding how exosome cofactors synergistically promote exosome function will provide us a better understanding of how the exosome complex precisely regulates its catalytic activities. As described here, cofactors play a major role in determining the substrate specificity of the nuclear and cytoplasmic exosome. Moreover, specific accessory domains, which are not involved in the catalytic function of the cofactor, are required for substrate targeting of the eukaryotic RNA exosome.

Differential Activity of the KRAS Oncogene by Method of Activation: Implications for Signaling and Therapeutic Intervention

Despite having been identified over thirty years ago and definitively established as having a critical role in driving tumor growth and predicting for resistance to therapy, the KRAS oncogene remains a target in cancer for which there is no effective treatment. KRas is activated b y mutations at a few sites, primarily amino acid substitutions at codon 12 which promote a constitutively active state. I have found that different amino acid substitutions at codon 12 can activate different KRas downstream signaling pathways, determine clonogenic growth potential and determine patient response to molecularly targeted therapies. Computer modeling of the KRas structure shows that different amino acids substituted at the codon 12 position influences how KRas interacts with its effecters. In the absence of a direct inhibitor of mutant KRas several agents have recently entered clinical trials alone and in combination directly targeting two of the common downstream effecter pathways of KRas, namely the Mapk pathway and the Akt pathway. These inhibitors were evaluated for efficacy against different KRAS activating mutations. An isogenic panel of colorectal cells with wild type KRas replaced with KRas G12C, G12D, or G12V at the endogenous loci differed in sensitivity to Mek and Akt inhibition. In contrast, screening was performed in a broad panel of lung cell lines alone and no correlation was seen between types of activating KRAS mutation due to concurrent oncogenic lesions. To find a new method to inhibit KRAS driven tumors, siRNA screens were performed in isogenic lines with and without active KRas. The knockdown of CNKSR1 (CNK1) showed selective growth inhibition in cells with an oncogenic KRAS. The deletion of CNK1 reduces expression of mitotic cell cycle proteins and arrests cells with active KRas in the G1 phase of the cell cycle similar to the deletion of an activated KRas regardless of activating substitution. CNK1 has a PH domain responsible for localizing it to membrane lipids making KRas potentially amenable to inhibition with small molecules. The work has identified a series of small molecules capable of binding to this PH domain and inhibiting CNK1 facilitated KRas signaling.

Pathway Semantics: An Algebraic Data Driven Algorithm to Generate Hypotheses about Molecular Patterns Underlying Disease Progression

The overarching goal of the Pathway Semantics Algorithm (PSA) is to improve the in silico identification of clinically useful hypotheses about molecular patterns in disease progression. By framing biomedical questions within a variety of matrix representations, PSA has the flexibility to analyze combined quantitative and qualitative data over a wide range of stratifications. The resulting hypothetical answers can then move to in vitro and in vivo verification, research assay optimization, clinical validation, and commercialization. Herein PSA is shown to generate novel hypotheses about the significant biological pathways in two disease domains: shock / trauma and hemophilia A, and validated experimentally in the latter. The PSA matrix algebra approach identified differential molecular patterns in biological networks over time and outcome that would not be easily found through direct assays, literature or database searches. In this dissertation, Chapter 1 provides a broad overview of the background and motivation for the study, followed by Chapter 2 with a literature review of relevant computational methods. Chapters 3 and 4 describe PSA for node and edge analysis respectively, and apply the method to disease progression in shock / trauma. Chapter 5 demonstrates the application of PSA to hemophilia A and the validation with experimental results. The work is summarized in Chapter 6, followed by extensive references and an Appendix with additional material.

Modeling Techniques for the High-Resolution Interpretation of Cryo-Electron Microscopy Reconstructions

Essential biological processes are governed by organized, dynamic interactions between multiple biomolecular systems. Complexes are thus formed to enable the biological function and get dissembled as the process is completed. Examples of such processes include the translation of the messenger RNA into protein by the ribosome, the folding of proteins by chaperonins or the entry of viruses in host cells. Understanding these fundamental processes by characterizing the molecular mechanisms that enable then, would allow the (better) design of therapies and drugs. Such molecular mechanisms may be revealed trough the structural elucidation of the biomolecular assemblies at the core of these processes. Various experimental techniques may be applied to investigate the molecular architecture of biomolecular assemblies. High-resolution techniques, such as X-ray crystallography, may solve the atomic structure of the system, but are typically constrained to biomolecules of reduced flexibility and dimensions. In particular, X-ray crystallography requires the sample to form a three dimensional (3D) crystal lattice which is technically di‑cult, if not impossible, to obtain, especially for large, dynamic systems. Often these techniques solve the structure of the different constituent components within the assembly, but encounter difficulties when investigating the entire system. On the other hand, imaging techniques, such as cryo-electron microscopy (cryo-EM), are able to depict large systems in near-native environment, without requiring the formation of crystals. The structures solved by cryo-EM cover a wide range of resolutions, from very low level of detail where only the overall shape of the system is visible, to high-resolution that approach, but not yet reach, atomic level of detail. In this dissertation, several modeling methods are introduced to either integrate cryo-EM datasets with structural data from X-ray crystallography, or to directly interpret the cryo-EM reconstruction. Such computational techniques were developed with the goal of creating an atomic model for the cryo-EM data. The low-resolution reconstructions lack the level of detail to permit a direct atomic interpretation, i.e. one cannot reliably locate the atoms or amino-acid residues within the structure obtained by cryo-EM. Thereby one needs to consider additional information, for example, structural data from other sources such as X-ray crystallography, in order to enable such a high-resolution interpretation. Modeling techniques are thus developed to integrate the structural data from the different biophysical sources, examples including the work described in the manuscript I and II of this dissertation. At intermediate and high-resolution, cryo-EM reconstructions depict consistent 3D folds such as tubular features which in general correspond to alpha-helices. Such features can be annotated and later on used to build the atomic model of the system, see manuscript III as alternative. Three manuscripts are presented as part of the PhD dissertation, each introducing a computational technique that facilitates the interpretation of cryo-EM reconstructions. The first manuscript is an application paper that describes a heuristics to generate the atomic model for the protein envelope of the Rift Valley fever virus. The second manuscript introduces the evolutionary tabu search strategies to enable the integration of multiple component atomic structures with the cryo-EM map of their assembly. Finally, the third manuscript develops further the latter technique and apply it to annotate consistent 3D patterns in intermediate-resolution cryo-EM reconstructions. The first manuscript, titled An assembly model for Rift Valley fever virus, was submitted for publication in the Journal of Molecular Biology. The cryo-EM structure of the Rift Valley fever virus was previously solved at 27Å-resolution by Dr. Freiberg and collaborators. Such reconstruction shows the overall shape of the virus envelope, yet the reduced level of detail prevents the direct atomic interpretation. High-resolution structures are not yet available for the entire virus nor for the two different component glycoproteins that form its envelope. However, homology models may be generated for these glycoproteins based on similar structures that are available at atomic resolutions. The manuscript presents the steps required to identify an atomic model of the entire virus envelope, based on the low-resolution cryo-EM map of the envelope and the homology models of the two glycoproteins. Starting with the results of the exhaustive search to place the two glycoproteins, the model is built iterative by running multiple multi-body refinements to hierarchically generate models for the different regions of the envelope. The generated atomic model is supported by prior knowledge regarding virus biology and contains valuable information about the molecular architecture of the system. It provides the basis for further investigations seeking to reveal different processes in which the virus is involved such as assembly or fusion. The second manuscript was recently published in the of Journal of Structural Biology (doi:10.1016/j.jsb.2009.12.028) under the title Evolutionary tabu search strategies for the simultaneous registration of multiple atomic structures in cryo-EM reconstructions. This manuscript introduces the evolutionary tabu search strategies applied to enable a multi-body registration. This technique is a hybrid approach that combines a genetic algorithm with a tabu search strategy to promote the proper exploration of the high-dimensional search space. Similar to the Rift Valley fever virus, it is common that the structure of a large multi-component assembly is available at low-resolution from cryo-EM, while high-resolution structures are solved for the different components but lack for the entire system. Evolutionary tabu search strategies enable the building of an atomic model for the entire system by considering simultaneously the different components. Such registration indirectly introduces spatial constrains as all components need to be placed within the assembly, enabling the proper docked in the low-resolution map of the entire assembly. Along with the method description, the manuscript covers the validation, presenting the benefit of the technique in both synthetic and experimental test cases. Such approach successfully docked multiple components up to resolutions of 40Å. The third manuscript is entitled Evolutionary Bidirectional Expansion for the Annotation of Alpha Helices in Electron Cryo-Microscopy Reconstructions and was submitted for publication in the Journal of Structural Biology. The modeling approach described in this manuscript applies the evolutionary tabu search strategies in combination with the bidirectional expansion to annotate secondary structure elements in intermediate resolution cryo-EM reconstructions. In particular, secondary structure elements such as alpha helices show consistent patterns in cryo-EM data, and are visible as rod-like patterns of high density. The evolutionary tabu search strategy is applied to identify the placement of the different alpha helices, while the bidirectional expansion characterizes their length and curvature. The manuscript presents the validation of the approach at resolutions ranging between 6 and 14Å, a level of detail where alpha helices are visible. Up to resolution of 12 Å, the method measures sensitivities between 70-100% as estimated in experimental test cases, i.e. 70-100% of the alpha-helices were correctly predicted in an automatic manner in the experimental data. The three manuscripts presented in this PhD dissertation cover different computation methods for the integration and interpretation of cryo-EM reconstructions. The methods were developed in the molecular modeling software Sculptor (http://sculptor.biomachina.org) and are available for the scientific community interested in the multi-resolution modeling of cryo-EM data. The work spans a wide range of resolution covering multi-body refinement and registration at low-resolution along with annotation of consistent patterns at high-resolution. Such methods are essential for the modeling of cryo-EM data, and may be applied in other fields where similar spatial problems are encountered, such as medical imaging.

Delayed Thrombus Resolution and Fibroproliferative Vascular Wound Healing From Deficiency of Type III Collagen: a Paradoxical Mechanism for Tissue Fragility

Vascular Ehlers-Danlos syndrome is a heritable disease of connective tissue caused by mutations in COL3A1, conferring a tissue deficiency of type III collagen. Cutaneous wounds heal poorly in these patients, and they are susceptible to spontaneous and catastrophic rupture of expansible hollow organs like the gut, uterus, and medium-sized to large arteries, which leads to premature death. Although the predisposition for organ rupture is often attributed to inherent tissue fragility, investigation of arteries from a haploinsufficient Col3a1 mouse model (Col3a1+/-) demonstrates that mutant arteries withstand even supraphysiologic pressures comparably to wild-type vessels. We hypothesize that injury that elicits occlusive thrombi instead unmasks defective thrombus resolution resulting from impaired production of type III collagen, which causes deranged remodeling of matrix, persistent inflammation, and dysregulated behavior by resident myofibroblasts, culminating in the development of penetrating neovascular channels that disrupt the mechanical integrity of the arterial wall. Vascular injury and thrombus formation following ligation of the carotid artery reveals an abnormal persistence and elevated burden of occlusive thrombi at 21 post-operative days in vessels from Col3a1+/- mice, as opposed to near complete resolution and formation of a patent and mature neointima in wild-type mice. At only 14 days, both groups harbor comparable burdens of resolving thrombi, but wild-type mice increase production of type III collagen in actively resolving tissues, while mutant mice do not. Rather, thrombi in mutant mice contain higher burdens of macrophages and proliferative myofibroblasts, which persist through 21 days while wild-type thrombi, inflammatory cells, and proliferation all regress. At the same time that increased macrophage burdens were observed at 14 and 21 days post ligation, the medial layer of mutant arterial walls concurrently harbored a significantly higher incidence of penetrating neovessels compared with those in wild-type mice. To assess whether limited type III collagen production alters myofibroblast behavior, fibroblasts from vEDS patients with COL3A1 missense mutations were seeded into three-dimensional fibrin gel constructs and stimulated with transforming growth factor-β1 to initiate myofibroblast differentiation. Although early signaling events occur similarly in all cell lines, late extracellular matrix- and mechanically-regulated events like transcriptional upregulation of type I and type III collagen secretion are delayed in mutant cultures, while transcription of genes encoding intracellular contractile machinery is increased. Sophisticated imaging of collagen synthesized de novo by resident myofibroblasts visualizes complex matrix reorganization by control cells but only meager remodeling by COL3A1 mutant cells, concordant with their compensatory contraction to maintain tension in the matrix. Finally, administration of immunosuppressive rapamycin to mice following carotid ligation sufficiently halts the initial inflammatory phase of thrombus resolution and fully prevents both myofibroblast migration into the thrombus and the differential development of neovessels between mutant and wild-type mice, suggesting that pathological defects in mutant arteries develop secondarily to myofibroblast dysfunction and chronic inflammatory stimulation, rather than as a manifestation of tissue fragility. Together these data establish evidence that pathological defects in the vessel wall architecture develop in mutant arteries as sequelae to abnormal healing and remodeling responses activated by arterial injury. Thus, these data support the hypothesis that events threatening the integrity of type III collagen-deficient vessels develop not as a result of inherent tissue weakness and fragility at baseline but instead as an episodic byproduct of abnormally persistent granulation tissue and fibroproliferative intravascular remodeling.