Bibliografische Beschreibung Knepper, Robert Darstellung des Verlaufs der Arteria palatina major unter dem Aspekt der Entnahme autologer Bindegewebstransplantate aus dem Gaumen Universität Leipzig, Dissertation 68 Seiten, 43 Literaturangaben, 42 Abbildungen, 12 Tabellen, 4 Anlagen Referat: Die sich stets weiterentwickelnden Verfahren zahnärztlicher Chirurgie bringen auch neue Anforderungen an den Operateur mit sich. Vor allem im Bereich der ästhetischen Korrektur von weichgewebigen Defekten im Bereich des Zahnhalteapparates gilt es, auf dem neuesten Stand zu sein, um moderne Zahnheilkunde anbieten zu können. Profunde Kenntnisse zur Topographie anatomischer Strukturen sind dabei für den Zahnarzt, den Spezialisten für Parodontologie und den Oralchirurgen unabdingbar. Um die dafür notwendige praktische Anleitung zu erhalten, können Mediziner am Institut für Anatomie der Universität Leipzig Kurse absolvieren, so z.B. auch angehende zahnärztliche Implantologen. Hierbei interessiert die Teilnehmer neben dem praktischen Vorgehen auch der Verlauf von Gefäßen und Nerven im Operationsgebiet. Anatomische Atlanten stellen die betreffenden Areale idealisiert und ohne Berücksichtigung der individuellen Variationsbreite dar. Zugänge, wie sie in der Klinik benötigt werden, finden sich selten. Aus diesem Grund wurde in der vorliegenden Arbeit an sechs vollständigen und 21 geteilten humanen Kopfpräparaten (alle Ethanol-Glycerin fixiert) die A. palatina major präpariert und der klinischen Situation gegenübergestellt. Diese Arterie wurde gewählt, da ihr Versorgungsgebiet – der Gaumen – die bevorzugte Spenderregion autologer Bindegewebstransplantate ist. Dadurch wird die Lücke der oft unzureichenden Verknüpfung der klinischen Situation mit dem anatomischen Korrelat geschlossen. Des Weiteren wurde an diesen Präparaten eine in der Literatur als sicher beschriebene Entnahmestelle vermessen und die Werte den bekannten gegenübergestellt. Um diese Messwerte beurteilen zu können, wurde außerdem die Frage gestellt, inwieweit die Fixierung der Präparate deren Beschaffenheit verändert. Dies wurde in einem Versuch mit dentalen Volumentomogrammen von frisch angefertigten und anschließend fixierten Präparaten untersucht. Auf Grund der Tatsache, dass Humanpräparate nur für einen begrenzten Zeitraum zur Verfügung stehen, ist ein wesentlicher Aspekt dieser makroskopisch-anatomischen Arbeit die fotografische Dokumentation der präparierten Gefäßstrecken, bei der ästhetische u.a. Gesichtspunkte beachtet werden müssen. Da dies auch für andere makroskopische Arbeiten am Institut für Anatomie von Interesse ist, wurde dieser Teil so ausführlich beschrieben, dass er als Anleitung dienen kann.:Inhaltsverzeichnis Abkürzungen VI 1. Einleitung 1 1.1. Problemstellung 1 1.2. Anatomische Grundlagen 3 1.2.1. Embryologische Entwicklung 3 1.2.2. Topographie 4 1.2.3. Mikroskopische Anatomie 7 1.3. Entnahme autologer Gewebstransplantate in der zahnärztlichen Chirurgie 7 1.3.1. Physiologie der freien Gewebstransplantation 7 1.3.2. Spenderregionen 9 1.3.3. Fallbeispiel 10 1.3.4. Verletzung der A. palatina major und Blutungskontrolle 11 2. Material und Methode 12 2.1. Untersuchungsmaterial 12 2.1.1. Wesentliche Angaben zum vorliegenden Material 12 2.1.2. Ethanol-Glycerin Fixierung 15 2.2. Präparationstechniken 16 2.3. Messmethoden 20 2.4. Statistische Auswertung 22 2.5. Einfluss der Fixierungsmethode auf das Präparationsmaterials 22 2.5.1. Materialveränderung durch die Fixierung 22 2.5.2. Versuchsaufbau 23 2.5.3. Dentale Volumentomografie 28 2.5.4. Verwendeter Dentaler Volumentomograph 29 2.5.5. DVT-Auswertungssoftware und Messmethode 30 2.6. Fotografische Dokumentation und Nachbearbeitung 32 2.6.1. Verwendete Hardware 32 2.6.2. Verwendete Software 33 2.6.3. Theoretische Grundlagen 35 2.6.4. Durchführung 38 2.6.5. Nachbearbeitung der Bilder 39 3. Ergebnisse 41 3.1. Vergleich zwischen klinischer Situation und anatomischem Präparat 42 3.2. Vermessung der Lage der A. palatina major 50 3.3. Verhalten von Weichgewebe unter dem Einfluss verschiedener Fixantien 52 3.4. Ergebnisse der Fotodokumentationen anatomischer Präparate 56 3.5. Fotografische Darstellung präformierter Körperhöhlen 58 4. Diskussion 60 4.1. Vergleich der Messergebnisse zur Lage der A. palatina major 60 4.2. Schlussfolgerungen aus dem DVT-Versuch 61 4.2.1. Vergleich der Messdaten für Kochen 61 4.2.2. Einfluss von Fixantien auf Weichgewebe 62 4.2.3. Aussagekraft der im DVT-Versuch ermittelten Werte 62 4.3. Kritische Einschätzung des Materials und der Methoden 63 4.3.1. Veränderungen der Anatomie unter Berücksichtigung der Präparation 63 4.3.2. Aussagekraft bezüglich Alter und Geschlecht der Spender 63 4.3.3. Kritische Einschätzung der angewandten Methoden 64 4.4. Weiterverwendung des Materials und der Erkenntnisse 65 4.4.1. Weiterverwendung Bildtafeln 65 4.4.2. Weiterverwendung DVT Versuchsaufbau 66 4.4.3. Weiterverwendung fotografische Anleitung 66 5. Zusammenfassung 67 6. Verzeichnis der Abbildungen 69 7. Verzeichnis der Tabellen 71 8. Literaturverzeichnis 72 9. Danksagung 76 10. Erklärung eigenständige Abfassung 77 11. Lebenslauf 78 Anlagen 79

Les opérations de hernies abdominales sont l’une des chirurgies les plus répandues dans le monde. Pourtant, malgré des progrès considérables en particulier dans le développement des textiles prosthétiques pour consolider la paroi abdominale, le taux de récurrence reste très élevé. Il apparaît donc nécessaire de développer des modèles numériques patient-spécifiques de la paroi abdominale afin de mesurer puis améliorer l’impact des solutions de soins. Les tissus assurant la cohésion et la stabilité de la paroi abdominale sont les gaines rectusiennes antérieure et postérieure et la ligne blanche, qui sont des tissus conjonctifs. Leur structure particulière, composée d’un entremêlement de fibres de collagène et d’élastine sont au cœur de cette étude.Ce mémoire a permis de mettre en évidence la relation entre architecture microscopique et comportement macroscopique des tissus fibreux. Grâce à des campagnes expérimentales combinant essais mécaniques et observations microscopiques, la structure des tissus a pu être identifiée et corrélée aux paramètres mécaniques. Des outils d’analyse d’images tridimensionnelles ont été développés afin d’estimer automatiquement l’anisotropie d’une texture. Les informations recueillies ont alors menées au développement d’un modèle constitutif anisotrope hypo-paramétré. Basé sur une description tridimensionnelle du réseau fibrillaire, le modèle a été écrit de manière à décorréler les paramètres liés à la structure et ceux relatifs à la nature même du matériau. En considérant les paramètres matériau similaires pour tous les individus, le modèle a démontré sa capacité à prédire le comportement mécanique à partir d’informations texturales / Abdominal hernia operations are one of the most common surgeries in the world. However, despite considerable progress, particularly in the development of prosthetic textiles to strengthen the abdominal wall, the recurrence rate remains very high. It therefore appears necessary to develop patient-specific numerical models of the abdominal wall in order to measure and improve the impact of care solutions. The tissues that ensure the cohesion and stability of the abdominal wall are the anterior and posterior rectus sheaths and the linea alba, which are connective tissues. Their particular structure, composed of an intertwining of collagen and elastin fibers, is at the heart of this study. This thesis highlighted the relationship between microscopic architecture and macroscopic behaviour of fibrous tissues. Thanks to experimental campaigns combining mechanical tests and microscopic observations, the structure of tissues has been identified and correlated to mechanical parameters. Three-dimensional image analysis tools have been developed to automatically estimate the anisotropy of a texture. The collected information then led to the development of a hypo-parameterized anisotropic constitutive model. Based on a three-dimensional description of the fibrillary network, the model was written in such a way as to uncorrelate the parameters related to the structure and those related to the nature of the material. By considering similar material parameters for every individual, the model demonstrated its ability to predict mechanical behaviour based on textural information

Despite the central role of IFNγ in vitiligo pathogenesis, systemic IFNγ neutralization is an impractical treatment option due to strong immunosuppression. However, most vitiligo patients present with less than 20% affected body surface area, which provides an opportunity for localized treatments that avoid systemic side effects. After identifying keratinocytes as key cells that amplify IFNγ signaling during vitiligo, I hypothesized that tethering an IFNγ neutralizing antibody to keratinocytes would limit anti-IFNγ effects to the treated skin for the localized treatment. To that end, I developed a bispecific antibody (BsAb) capable of blocking IFNγ signaling while binding to desmoglein expressed by keratinocytes. I characterized the effect of the BsAb in vitro, ex vivo, and in a mouse model of vitiligo. SPECT/CT biodistribution and serum assays after local footpad injection revealed that the BsAb had improved skin retention, faster elimination from the blood, and less systemic IFNγ inhibition than the non-tethered version. Furthermore, the BsAb conferred localized protection almost exclusively to the treated footpad during vitiligo that was not possible by local injection of the non-tethered anti-IFNγ antibody. Thus, keratinocyte-tethering proved effective while significantly diminishing off-tissue effects of IFNγ blockade, offering a new treatment strategy for localized skin diseases, including vitiligo.

The 21st century brought about a dramatic increase in knowledge about genetic and molecular profiles of cancer. This information has validated the complexity of tumor cells and increased awareness of “nodal proteins”, but has yet to advance the development of rational targeted cancer therapeutics. Nodal proteins are critical cellular proteins that collect biological inputs and distribute the information across diverse biological processes. Survivin acts as a nodal protein by interfacing the multiple signals involved in mitosis and apoptosis and functionally integrate proliferation, cell death, and cellular homeostasis. By characterizing survivin as a target of both Type 1 Insulin-like Growth Factor (IGF-1) and Notch developmental signaling, we contribute to the paradigm of survivin as a nodal protein. The two signaling systems, Notch and IGF-1, regulate survivin by two independent mechanisms. Notch activation induces survivin transcription preferentially in basal breast cancer, a breast cancer subtype with poor prognosis and lack of molecular therapies. Activated Notch binds the transcription factor RBP-Jк and drives transcription from the survivin promoter. Notch mediated survivin expression increases cell cycle kinetics promoting tumor proliferation. Inhibition of Notch in a breast xenograft model reduced tumor growth and systemic metastasis. On the other hand, IGF-1 signaling drives survivin protein translation in prostate cancer cells. Binding of IGF-1 to its receptor activates downstream kinases, mammalian target of rapamycin (mTOR) and p70 S6 protein kinase (p70S6K), which modulates survivin mRNA translation to increase the apoptotic threshold. The multiple roles of survivin in tumorigenesis implicate survivin as a rational target for the “next generation” of cancer therapeutics.

Remarkable progress has been made in the development of effective treatments for patients with rheumatoid arthritis (RA). To ensure that a patient is optimally responding to treatment, consistent monitoring of disease activity is recommended. Established composite and individual disease activity measures often cannot be computed due to missing laboratory values. Simplified measures that can be calculated without a lab value have been developed and previous studies have validated these new measures, yet differences in their performance compared with established measures remain. Therefore, the goal of my doctoral research was to examine and evaluate disease activity and composite measures to facilitate monitoring of response in clinical care settings and inclusion of patients with missing laboratory values in epidemiological research. In the first study, the validity of two composite measures, the Clinical Disease Activity Index (CDAI) and the Disease Activity Score with 28 joint count (DAS28) was examined and both were significantly associated with a rheumatologist’s decision to change therapy (CDAI OR=1.58; 95% CI: 1.42, 1.76) (DAS28 OR=1.34; 95% CI 1.27,1.56). However, further evaluation using receiver operating characteristic (ROC) analysis found that they were not strong predictors of physician decisions to change therapy (AUC=0.75, 0.76, respectively). Thus, they should not be used to guide treatment decisions in the clinic. Two measures of disease activity, erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) are often not measured and impede the computation of composite measures of disease activity. In the second study, significant factors which may predict the measurement of the ESR and CRP were identified and included physician and clinical variables but no quantitative disease activity measures. Thus the suitability of the ESR and CRP as measures of disease activity is suspect. In the final study, I created a new composite measure, the modified disease activity score with 28 joint count (mDAS28), by replacing the laboratory value in the DAS28. The mDAS28 was then validated by comparing its performance with the DAS28. The measures were strongly correlated (r=0.87), and strong agreement was found between the two measures when categorizing patients to levels of disease activity (ĸ=0.77) and treatment response (ĸ=0.73). Therefore, the mDAS28 could be used in place of the DAS28 when laboratory values needed to compute the DAS28 are missing. In summary, I found that the CDAI and DAS28 were not strong predictors of the rheumatologist’s decision to change therapy. I also found that the variability in the measurement of ESR and CRP was not associated with disease activity. I was able to modify the DAS28 by replacing the laboratory measure and create a new simplified measure, the mDAS28. I also validated the mDAS28 for use in the clinic and in epidemiological research when the DAS28 is unavailable.

The development and survival of multicellular organisms depends upon the ability of cells to move. Embryogenesis, immune surveillance, wound healing, and metastatic disease are all processes that necessitate effective cellular locomotion. Central to the process of cell motility is the family of integrins, transmembrane cell surface receptors that mediate stable adhesions between cells and their extracellular environment. Many human diseases are associated with aberrant integrin function. Carcinoma cells in particular can hijack integrins, harnessing their mechanical and signaling potential to propagate cell invasion and metastatic disease, one example being integrin α6β4. This integrin, often referred to simply as β4, is defined as an adhesion receptor for the laminin family of extracellular matrix proteins. The role of integrin β4 in potentiating carcinoma invasion is well established, during which it serves both a mechanical and signaling function. miRNAs are short non-coding RNAs that regulate gene expression posttranscriptionally, and data describing the role of extracellular stimuli in governing their expression patterns are sparse. This observation coupled to the increasingly significant role of miRNAs in tumorigenesis prompted us to examine their function as downstream effectors of β4, an integrin closely linked to aggressive disease in breast carcinoma. The work presented in this dissertation documents the first example that integrin expression correlates with specific miRNA patterns. Moreover, integrin β4 status in vitro and in vivo is associated with decreased expression of distinct miRNA families in breast cancer, namely miR-25/32/92abc/363/363-3p/367 and miR-99ab/100, with purported roles in cell motility. Another miRNA, miR-29a, is significantly downregulated in response to de novo expression of β4 in a breast carcinoma cell line, and β4-mediated repression of the miRNA is required for invasion. Another major conclusion of this study is that β4 integrin expression and ligation can regulate the expression of SPARC in breast carcinoma cells. These data reveal distinct mechanisms by which β4 promotes SPARC expression, involving both a miR-29a-mediated process and a TOR-dependent translational mechanism. Our observations establish a link between miRNA expression patterns and cell motility downstream of β4 in the context of breast cancer, and uncover a novel effector of β4-mediated invasion.

Despite the importance of integrins in epithelial cell biology surprisingly little is known about their regulation. It is known that they form hemidesmosomes (HDs), are actively involved in cell contacts during cell migration/invasion, and are key signaling molecules for survival and growth. However, there has been a distinct lack of understanding about what controls the dynamic integrin localization during cell activation and movement. Growth factors, such as EGF, are elevated during wound healing and carcinoma invasion leading to phosphorylation of ITGβ4 and the disassembly of the HD and mobilization of ITGβ4 to actin-rich protrusions. More recently the phosphorylation of a novel site on ITGβ4 (S1424) was found to be distinctly enriched on the trailing edge of migrating cells, suggesting a possible mechanism for the dissociation of ITGβ4 from HDs. Arrestin family member proteins are involved in the regulation of cell surface proteins and vesicular trafficking. In this study, we find that over-expression of arrestin family member ARRDC3 causes internalization and proteosome-dependent degradation of ITGβ4, while decreased levels of ARRDC3 stabilizes ITGβ4 levels. These results lead us to a new mechanism of ITGβ4 internalization, trafficking and degradation. During migration, ARRDC3 co-localizes with ITGβ4 on the lagging edge of cells but has a distinct distribution on the leading edge of cells. Additional immuno co-precipitation experiments demonstrate that ARRDC3 preferentially binds to ITGβ4 when phosphorylated on S1424. Using confocal microscopy, we show that the expression pattern of ARRDC3 on the lagging edge of a migrating cell is identical to the expression pattern of ITGβ4-pS1424. We demonstrate that ARRDC3 expression represses cell proliferation, migration, invasion, growth in soft agar and tumorigenicity. Collectively, our data reveals that ARRDC3 is a negative regulator of β4 integrin and demonstrates how this new pathway impacts biologic processes in stem cell and cancer biology. Additionally, as ARRDC3 is highly expressed in several tissues and conserved across species, our results are likely to be translated to other models.

As humans, we inhabit an environment shared with many microorganisms, some of which are harmless or beneficial, and others which represent a threat to our health. A complex network of organs, cells and their protein products form our bodies’ immune system, tasked with detecting these potentially harmful agents and eliminating them. This same system also serves to detect changes in the healthy balance of normal functions in the body, and for repairing tissue damage caused by injury. Immune recognition of nucleic acids, DNA and RNA, is one way that the body detects invading pathogens and initiates tissue repair. A number of specialized receptor proteins have evolved to distinguish nucleic acids that represent “threats” from those involved in normal physiology. These proteins include members of the Toll-like receptor family and diverse types of cytosolic proteins, all of which reside within the confines of the cell. Few proteins on the cell surface have been clearly characterized to interact with nucleic acids in the extracellular environment. In this dissertation, I present collaborative work that identifies the receptor for advanced glycation end products (RAGE) as a cell surface receptor for nucleic acids and positions it as an important modulator of immune responses. Molecular dimers of RAGE interact with the sugar-phosphate backbones of nucleic acid ligands, allowing this receptor to recognize a variety of DNA and RNA molecules regardless of their nucleotide sequence. Expression of RAGE on cells promotes uptake of DNA and enhances subsequent responses that are dependent on the nucleic acid sensor Toll-like receptor 9. When mice deficient in RAGE are exposed to DNA in the lung, the predominant site of RAGE expression, they do not mount a typical early inflammatory response, suggesting that RAGE is important in generating immune responses to DNA in mammalian organisms. Further evidence suggests that RAGE interacts preferentially with multimolecular complexes that contain nucleic acids, and that these complexes may induce clustering of receptor dimers into larger multimeric structures. Taken together, the data reported here identify RAGE as an important cell surface receptor protein for nucleic acids, which is capable of modulating the intensity of immune responses to DNA and RNA. Understanding of and intervention in this recognition pathway hold therapeutic promise for diseases characterized by excessive responses to self nucleic acids, such as systemic lupus erythematosus, and for the pathology caused by chronic inflammatory responses to self and foreign nucleic acids.

Apurinic/apyrimidinic DNA repair endonuclease-1 (APE1), first recognized as an important DNA excision repair enzyme, is also known as Redox Factor-1 (Ref-1) involved in the activation of many nuclear transcription factors in both redox-dependent and independent manner. It has been well-documented that the overexpression of APE/Ref-1 contributes to the development of chemo-resistance and is associated with tumor progression in many human malignancies [1]. Our previous study in melanoma demonstrated that the development of novel inhibitors targeting the redox regulation domain of APE/Ref-1 is a promising strategy for melanoma treatment. To date, limited successes have been reported in developing novel APE/Ref-1 inhibitors for cancer treatment. Utilizing a structure-based approach, our study identified and characterized small molecular inhibitors of APE/Ref-1. First, N-terminally truncated APE/Ref-1 protein lacking the first 40 amino acid residues (∆40APE-1wt) was cloned into the pGEX-6P1 vector to express the GST-∆40APE-1wtprotein. After cleavage of GST-tag, the concentrated ∆40APE-1wt protein was subjected to protein crystallization study. We have successfully diffracted ∆40APE-1wt crystals and collected data with a resolution of 1.57Å. The crystal structure was further determined by molecular replacement in Molrep using the already available human APE-1 structure (PDB: 5CFG). For the first time, we observed the dimerization of APE/Ref-1 protein formed under oxidative conditions, which may contribute to the redox regulation of APE/Ref-1. Such structural transformation of APE/Ref-1 protein under distinct redox conditions may pave the way for future drug development and optimization. The binding affinity of the candidate compounds with ∆40APE-1wt protein was also determined using Surface Plasmon Resonance (SPR), and the Ki values were analyzed. One of the potent inhibitors developed by our group by structure-based approach, exhibited promising anti-melanoma activities both in vitro and in vivo. Future studies on the structure-activity association are warranted.

Indoor tanning (IT) before the age of 35 increases one’s risk for melanoma by 75%, and epidemiological data show a 6.1% annual increase in the incidence of melanomas in white women younger than age 44 in the US. Population-based studies reveal that 15% of adolescents and 8% to 14% of their primary caregivers have engaged in IT in the past year. The compelling case for IT being a significant risk factor for melanoma, together with the high rates of IT in teen girls and their mothers, provided a strong rationale for conducting an antitanning intervention directed at mother-daughter dyads. This study evaluated a strategy designed to prevent skin cancer in adolescent girls by using mothers as change agents to effectively communicate the risks of IT and to encourage teens to avoid high risk IT behaviors. Mother-daughter dyads were recruited over the telephone, randomly assigned to the intervention or control group, and surveyed on IT risk constructs including tanning-specific knowledge and communication. Forty-two mother-daughter dyads completed baseline surveys in the summer of 2012. Mothers in the intervention group were given a handbook educating them on the dangers of IT and how to convey information about skin cancer prevention to their daughters and encouraged to talk with their daughters about the issues covered in the handbook over a 1-month period. Participants completed follow-up assessments in October 2012 and January 2013. Among teens, past 3-month IT frequency, intentions, and willingness decreased in intervention group teens, while intentions and willingness increased among control teens. Intervention teens exhibited lower IT attitudes and higher levels of perceived susceptibility to appearance damage and health effects from IT when compared to control teens. Intervention teens reported higher levels of maternal monitoring and lower levels of maternal permissiveness toward IT. Qualitative data indicated mothers responded positively to the handbook, and it encouraged tanning-specific discussions with their daughters. Mothers provided suggestions on how to improve the handbook, that once incorporated, should lead to improved intervention efficacy. Overall, study results indicated this intervention strategy is feasible, as mothers did communicate with their teens and were able to convey the antitanning messages.