Einleitung/ Zielstellung Zusätzlich zur Gammastrahlung emittiert 99mTc ca. 5 niederenergetische Auger-Elektronen mit Reichweiten von wenigen Nanometern im Gewebe. Diese haben für die nuklear-medizinische Diagnostik keine Bedeutung. Es wird jedoch über eine therapeutische Nutzung diskutiert, wofür eine Anreicherung der Auger-Elektronen-Emitter in einem strahlensensitiven Zellkompartiment erforderlich ist. Ziel der Arbeit war es, verschiedene [99mTc]Tc-Radiopharmaka hinsichtlich ihres Uptakeverhaltens, der subzellulärer Verteilung und des Retentionsverhaltens in vitro zu untersuchen, sowie die mutmaßlich durch den Auger-Effekt hervorgerufene Radiotoxizität der [99mTc]Tc-markierten Radiopharmaka zu vergleichen und die gewonnenen Ergebnisse in Hinblick auf potentielle extranukleäre strahlensensitive Targets zu interpretieren. Material und Methode Durchgeführt wurden die Versuche im ersten Abschnitt der Arbeit an Natrium-Iodid-Symporter (NIS)-positiven FRTL-5-Schilddrüsenzellen. Von [99mTc] Pertechnetat ([99mTc]TcO4-), [99mTc]TcO4- nach Vorinkubation von Perchlorat ([99mTc]TcO4-/ ClO4-), [99mTc]Tc-Hexakis-2-Methoxyisobutylisonitril ([99mTc]Tc-MIBI), [99mTc]Tc-Hexamethyl-Propylenaminoxim ([99mTc]Tc-HMPAO) und [99mTc]TcO4- nach Vorinkubation von Zinn-Pyrophosphat (Sn- PYP/ [99mTc]TcO4-) wurden die intrazelluläre Radio¬nuklid¬aufnahme und die subzelluläre Verteilung untersucht. Basierend auf den Ergebnissen dieser Versuche wurde die mittlere absorbierte Zellkerndosis kalkuliert. Zur Beurteilung der strahlenbiologischen Wirkung wurde das klonogene Zellüberleben mit der Anzahl residualer gH2AX-Foci (DNA-Schaden) verglichen und die Wirkung der [99mTc]Tc Tracer auf den Zellzyklus von FRTL-5-Zellen untersucht. Im zweiten Abschnitt der Arbeit wurde an EGFR-positiven A431-Zellen die radiotoxische Wirkung in Abhängigkeit von der intra¬zellulären Lokalisation von [99mTc]Alexa(488)-C225-Cyclooctin-Dpa-Tc(CO)3 ([99mTc]Tc-C225), [99mTc]Tc-HMPAO und [99mTc]TcO4- auf das klonogene Zellüberleben untersucht. Ergebnisse und Diskussion Aufgrund verschiedener Uptakemechanismen zeigte jedes der untersuchten [99mTc]Tc-Radiopharmaka Unterschiede im zeitlichen Verlauf der Uptakekinetik. Durch Blockierung des NIS durch ClO4- konnte eine intrazelluläre Aufnahme von [99mTc]TcO4- verhindert werden, wogegen durch Vorinkubation mit Sn-PYP die zelluläre Aufnahme von [99mTc]TcO4- um das 22-fache gesteigert wurde. [99mTc]Tc-MIBI und [99mTc]Tc-HMPAO wurden aufgrund ihrer lipophilen Eigenschaften unabhängig vom NIS passiv in die Zelle transportiert. Die Untersuchung der intrazellulären Verteilung ergab für [99mTc]Tc-HMPAO und Sn-PYP/ [99mTc]TcO4- eine vergleichbar hohe Anreicherung in der Membran/Organellen-Fraktion sowie in der Zellkernfraktion. Von [99mTc]TcO4- und [99mTc]Tc-MIBI wurde die Hauptaktivität in der Zytosol-Fraktion und nur geringe Anteile in der Membran/Organellen-Fraktion sowie in der Zellkernfraktion nachgewiesen. In guter Übereinstimmung zur subzellulären Verteilung zeigten Sn-PYP/ [99mTc]TcO4- und [99mTc]Tc-HMPAO eine fast vollständige, hingegen [99mTc]Tc-MIBI und [99mTc]TcO4- nur eine geringe Retention. Aufgrund der genannten Unterschiede wurde bei gleicher inkubierter Aktivitätskonzentration folgende Reihenfolge der resultierenden Zellkerndosis ermittelt: [99mTc]TcO4- < [99mTc]Tc-MIBI < [99mTc]Tc-HMPAO < Sn-PYP/ [99mTc]TcO4-. [99mTc]TcO4- und [99mTc]Tc-HMPAO zeigten eine ähnliche Wirkung auf das klonogene Zellüberleben und auf den Zellzyklus. Jedoch bewirken sie eine wesentlich stärkere Reduzierung des Überlebens und einen stärkeren G2-Arrest als [99mTc]Tc-MIBI und Sn-PYP/ [99mTc]TcO4-, wobei [99mTc]Tc-MIBI bei allen drei untersuchten biologischen Endpunkten die geringste Wirkung zeigte. Bei einer vergleichbaren Reduktion des Zellüberlebens von [99mTc]TcO4- und [99mTc]Tc-HMPAO induzierte [99mTc]Tc-HMPAO lediglich die Hälfte der gH2AX-Foci im Vergleich zu [99mTc]TcO4-. Die trotz geringerem DNA-Schaden vergleichbare radiotoxische Wirkung zeigte, dass das klonogene Zellüberleben nicht allein vom DNA-Schaden abhängt. Daraus folgt, dass es außer der Kern-DNA noch weitere strahlensensitive Kompartimente gibt, die durch [99mTc]Tc-HMPAO stärker geschädigt wurden als von den anderen untersuchten [99mTc]Tc Tracern. Ein mögliches extranukleäres strahlensensitives Target ist die Zellmembran, so dass im zweiten Teil der Arbeit zur Überprüfung der Radiosensitivität der Zellmembran die radiotoxische Wirkung von [99mTc]Tc-C225 an EGFR-positiven A431-Zellen untersucht wurde. [99mTc]Tc-C225 wurde über den EGFR und [99mTc]Tc-HMPAO aufgrund seiner Lipophilie durch Diffusion intrazellulär aufgenommen. [99mTc]TcO4- dagegen zeigte keine intrazelluläre Aufnahme in die NIS-negativen Zellen und wurde als Referenz für eine extrazelluläre Bestrahlung verwendet. [99mTc]Tc-C225 wies nach einstündiger Inkubation eine Membranbindung von lediglich 10 % auf, die im Laufe von 24 h auf 1,9 % absank. Dies zeigte, dass [99mTc]Tc-C225 rasch in den A431-Zellen internalisiert wurde und dass nur bei sehr kurzen Inkubationszeiten von einer spezifischen Zellmembranmarkierung gesprochen werden kann. [99mTc]Tc-HMPAO ging keine Bindung an die Zellmembran ein. Weiterhin wurde bei der Inkubation steigender Aktivitäts- und Antikörperkonzentrationen von [99mTc]Tc C225 eine Sättigung des EGFR beobachtet, woraus eine wesentlich geringere Zellkerndosis als bei Inkubation von [99mTc]Tc-HMPAO resultierte. Im Vergleich des klonogenen Zellüberlebens zeigten [99mTc]Tc-C225 und [99mTc]Tc-HMPAO bei gleicher Zellkerndosis keine Unterschiede in der radiobiologischen Wirkung. Somit konnte lediglich eine Verstärkung der radiotoxischen Effekte von [99mTc]Tc-C225 an A431-Zellen im Vergleich zur ausschließlich extrazellulären Verteilung von [99mTc]TcO4- gezeigt werden. Schlussfolgerung Die Untersuchung der radiotoxischen Wirkung von [99mTc]Tc-C225 ermöglichte bei den angewendeten Versuchsbedingungen keine Rückschlüsse auf die Strahlensensitivität der Zellmembran. Weiterführende Arbeiten zur Entwicklung eines 99mTc-markierbaren spezifischen Membranmarkers wären notwendig, um klären zu können, ob die Zellmembran ein ähnlich strahlensensitives Target wie die nukleäre DNA ist. Dosimetrische Betrachtungen an den als Modellsystemen dienenden FRTL-5- und A431-Zellen deuten darauf hin, dass aufgrund ungenügender Anreicherung eine therapeutische Wirkung der Auger-Elektronen im Tumorgewebe eher unrealistisch ist. Damit sollte aus gegenwärtiger Sicht die klinische Anwendung von 99mTc auf den diagnostischen Einsatz beschränkt bleiben. Jedoch könnte 99mTc als Auger-Elektronen-Emitter bei spezifischer Anreicherung in definierten Zellkompartimenten als Nano-Tool zur Erforschung der Strahlensensitivität einzelner Zellbestandteile eingesetzt werden. / Introduction In addition to gamma radiation, 99mTc emits approximately 5 low energy Auger and internal conversion electrons per decay, resulting in high ionization density proximal to the radionuclide’s decay position. Low-energy Auger electrons with path lengths of only nanometers cannot be utilized for diagnostic procedures; however, they have frequently been discussed for therapeutic applications. To achieve a radiobiological effect, an intracellular accumulation and distribution in relevant cell compartments of the Auger electron emitter is required. Aim The aim of the thesis was the comparison of different [99mTc]Tc-labeled compounds concerning their intracellular uptake, subcellular distribution and retention in vitro. Furthermore the radiotoxicity caused by the Auger effect has to be investigated. Material and Methods The intracellular radionuclide uptake, subcellular distribution (ProteoExtract®-Kit) and retention of [99mTc] pertechnetate ([99mTc]TcO4-), [99mTc]TcO4- after pre-incubation of perchlorate ([99mTc]TcO4-/ClO4-), [99mTc]TcO4- after pre-incubation of stannous pyrophosphate ([99mTc]TcO4-/Sn-PYP), [99mTc]Tc-hexamethyl-propylene-aminoxime ([99mTc]Tc-HMPAO) and [99mTc]Tc-hexakis-2-methoxyisobutylisonitrile ([99mTc]Tc-MIBI) were quantified in sodium-iodide symporter (NIS)-positive rat thyroid FRTL-5 cells. Basing on these results the mean absorbed nucleus dose was calculated. Radiotoxicity was investigated using phosphorylated histone H2AX (gH2AX foci), clonogenic cell survival and cell cycle analyzes. Additionally the radiotoxicity of [99mTc]Alexa(488)-C225-Cyclooctin-Dpa-Tc(CO)3 ([99mTc]Tc-C225) was compared with the one of [99mTc]TcO4- and [99mTc]Tc -HMPAO depending on the subcellular distribution in EGFR-positive A431 cells. Results and Discussion For the analyzed [99mTc]Tc-labeled compounds we detected differences in the time courses of the uptake kinetics caused by different uptake mechanisms into the FRTL-5 cells. The radionuclide uptake of [99mTc]TcO4- was blocked in the presence of perchlorate and increased by a factor of approximately 22 after pre-incubation of Sn-PYP. The lipophilic complexes [99mTc]Tc-MIBI and [99mTc]Tc-HMPAO crossed the cell membrane through passive transport via diffusion. The compartmental analysis indicated that [99mTc]Tc-HMPAO and [99mTc]TcO4-/Sn-PYP revealed a comparable high uptake in the nucleus and in the membrane/organelle fraction. [99mTc]TcO4- and [99mTc]Tc-MIBI were preferentially distributed in the cytosol, with lower amounts of the accumulated activity in both the membranes/organelles and the nucleus compared with the other compounds. In good agreement with the subcellular distribution [99mTc]Tc-HMPAO, [99mTc]TcO4-/Sn-PYP showed a nearly complete retention and [99mTc]TcO4-, [99mTc]Tc-MIBI a low retention. Due to the differences mentioned above the following sequence of the calculated mean nucleus dose for identical activity concentrations was determined: [99mTc]TcO4- < [99mTc]Tc-MIBI < [99mTc]Tc-HMPAO < Sn PYP/ [99mTc]TcO4-. [99mTc]TcO4- and [99mTc]Tc-HMPAO caused a similar reduction of the cell survival and a dose dependent G2-arrest. [99mTc]Tc-MIBI and Sn-PYP/ [99mTc]TcO4- are both less radiotoxic in terms of the estimated nucleus dose compared with [99mTc]TcO4- and [99mTc]Tc-HMPAO. Despite the similar effect on the cell survival [99mTc]Tc-HMPAO induced only half of the residual gH2AX foci than [99mTc]TcO4-. These findings reveal that clonogenic cellular survival is not solely determined by the DNA-DSB response, which may suggest the involvement of extra-nuclear radiosensitive targets in cell inactivation. A possible extra-nuclear radiosensitive target is the cell membrane. That’s why the aim of the second part of the thesis is the investigation of the radiosensitivity of the cell membrane. Therefore the radiotoxic influence of [99mTc]Tc-C225 was analyzed at EGFR-positive A431 cells. [99mTc]Tc-C225 was taken up over the EGFR and the lipophilic [99mTc]Tc-HMPAO was transported via diffusion over the cell membrane. In contrast, [99mTc]TcO4- did not show any intracellular uptake into the NIS-negative cells and therefore was used as extracellular reference. An incubation of [99mTc]Tc-C225 for one hour resulted to a membrane binding of only 10 %, which was reduced to 1.9 % after 24 hours. This demonstrated a fast internalization into A431-cell. Therefore only in the case of a very short incubation time [99mTc]Tc-C225 leads to a specific targeting of the cell membrane. [99mTc]Tc-HMPAO did not bind to the cell membrane. Furthermore the incubation of increasing concentrations of activity and antibody resulted in a saturation of the EGFR, leading to a significant lower nucleus dose in comparison to the incubation of [99mTc]Tc-HMPAO. Concerning the clonogenic cell survival no differences in the radiotoxicity of [99mTc]Tc-C225 and [99mTc]Tc-HMPAO were observed for equal nucleus dose. Thus only an amplification of the radiotoxic effects of [99mTc]Tc-C225 in comparison to the extracellular distribution in A431 cells of 99mTc-pertechnetate was observed. Conclusion The investigation of the radiotoxic effect of [99mTc]Tc-C225 did not allow any conclusions about the radiosensitivity of the cell membrane under the given experimental conditions. For clarifying if the radiosensitivity of the cell membrane is comparable to the one of the nucleus DNA further experiments for the development of a [99mTc]Tc-labeled specific target for the cell membrane are necessary. On the basis of the dosimetric considerations of the FRTL-5 cells and A431 cells used as model systems it can be concluded that because of an insufficient accumulation a therapeutic radiotoxic effect of the Auger electrons is not realistic. Therefore the clinical use of 99mTc should be limited to the diagnostics. Nevertheless specific accumulated Auger electrons of 99mTc could be applied in the field of investigation as nano-tools for the subcellular analysis of radiotoxicity.

Photocontrol, the use of light-sensitive proteins to control events within living tissue, allows complex processes in higher organisms to be studied. The Halorhodospira halophila photoactive yellow protein (PYP) can be used to regulate transcription factor activity with blue light. Before any PYP-based system can probe complex processes in higher organisms, proof of functional expression in vivo is required. We linked d25 PYP to the C-terminus of blue fluorescent protein (BFP) and expressed variants of the fusion protein (BFPd25PYP) in E. coli and human embryonic kidney (HEK293) cells. Expression of BFPd25PYP in E. coli verified in vitro photoswitching. The fusion protein was successfully expressed in HEK293. Fluorescence studies of intact cells indicated chromophore uptake and incorporation into PYP in HEK293, while photoswitching of PYP was measured in protein isolated from HEK293. These findings are promising for the development of applications using PYP for in vivo mammalian photocontrol of biological events.

Photocontrol, the use of light-sensitive proteins to control events within living tissue, allows complex processes in higher organisms to be studied. The Halorhodospira halophila photoactive yellow protein (PYP) can be used to regulate transcription factor activity with blue light. Before any PYP-based system can probe complex processes in higher organisms, proof of functional expression in vivo is required. We linked d25 PYP to the C-terminus of blue fluorescent protein (BFP) and expressed variants of the fusion protein (BFPd25PYP) in E. coli and human embryonic kidney (HEK293) cells. Expression of BFPd25PYP in E. coli verified in vitro photoswitching. The fusion protein was successfully expressed in HEK293. Fluorescence studies of intact cells indicated chromophore uptake and incorporation into PYP in HEK293, while photoswitching of PYP was measured in protein isolated from HEK293. These findings are promising for the development of applications using PYP for in vivo mammalian photocontrol of biological events.

HIV prevention programs in schools are acknowledged as one of the best prospects for controlling the world HIV epidemic. Epidemiological evidence indicates that deaths world-wide from AIDS are yet to peak. Although HIV notifications and AIDS deaths in the total Australian population have decreased', there has been an increase in rates in the Australian Indigenous population. There is also some evidence of complacency in HIV prevention vigilance in Australia which indicates a need for continued focus on prevention programs. The knowledge levels, attitudes toward HIV risk, and risk-taking behaviours of young Australians place them at risk of exposure to HIV. Appropriate prevention programs can be delivered to these vulnerable young people through the school setting. Programs delivered in schools have been shown to have a positive effect and teachers are vital to the delivery of these education programs. Without appropriate training, however, teachers may not optimise the outcomes of these programs. While it would be desirable for teachers to be trained in HIV prevention education in pre-service training this has not been the case in Western Australia (WA). When there is not an opportunity for pre-service training, professional development programs can be implemented to provide additional training required by teachers. Traditionally this professional development has been provided through workshops. These face-to-face delivery methods, however, do not always adequately serve the needs of all teachers, and in particular the needs of teachers in rural and remote areas. In an attempt to address the needs of these teachers, alternate methods of professional development delivery may be appropriate. The aim of this study was to test an alternate method of delivery. / The study designed, disseminated and evaluated the implementation of a flexible learning professional development program for teachers of HIV education. The program was based on print-based distance learning, supported by a video and tutors. Five objectives were developed for the study. These objectives were: Objective One - To determine factors associated with teachers' enrolment in the Protect Yourself Program (PYP). Objective Two - To determine the association between factors related to entry characteristics, social integration, external attribution, academic integration and incompatibility and amount of PYP completed. Objective Three - To determine the association between amount of PYP completed and factors related to the teaching of HIV lessons. Objective Four - To examine the context in which intervention and comparison group teachers were operating for this study. Objective Five - To evaluate the process of teacher recruitment to PYP, satisfaction with the flexible learning methodology, satisfaction with the PYP materials and completion of PYP. A comprehensive theoretical framework was constructed to guide the development of the empirical study and the professional development program, as little evidence was found in the literature of similar empirically evaluated studies. This framework incorporated: Adult Learning Theory; the Model of Student Progress; the PRECEDEPROCEED Model; the Health Promoting Schools Framework; Diffusion of Innovation and the Communication Behaviour Change Model. The study was conducted in two parts. Firstly, an exploratory study was conducted which provided a basis upon which to implement the second, larger empirical study. / A quasi-experimental study design was implemented due to restrictions placed upon the study by the WA Department of Health, the funding agency. The study sample was made up of teachers from government and independent, primary and second schools in WA. In total, 126 teachers were recruited to the intervention group and enrolled in the professional development program, and 128 to the comparison group, who completed some of the evaluation instruments, but did not participate in a professional development program. The professional development intervention program incorporated four comprehensive work modules, which were delivered in print form. A video and tutorial support supplemented the print materials. To evaluate the professional development program, seven instruments were developed. From these instruments five categories of variables were created, namely demographic, contextual, teacher characteristics, process and dependent. These variables were developed as single item variables, scales or indices. Quantitative data were analysed using the Statistical Package for the Social Sciences and a combination of univariate, bivariate, and multivariate techniques (logistic regression and analysis of covariance) were conducted. Qualitative data were analysed for themes. A binary logistic regression was conducted to evaluate Objective One: to determine factors associated with teachers’ enrolment in PYP. The analysis identified four factors which were associated with enrolment in PYP. / The teachers most likely to enrol in PYP had no pre-service training in health education and did not consider themselves to be a specialist or coordinator of health education. The majority of program participants had been teaching health education for between three and seven years and displayed a high level of acceptance of the flexible learning methodology. Objective Two: to determine the association between factors related to entry characteristics, social integration, external attribution, academic integration and incompatibility and amount of PYP completed was evaluated using a nominal logistic regression analysis with the intervention group sample only. Completion of the PYP program by participants was related to circumstances which were often beyond the control of the program, such as events occurring in a teacher’s personal life. However, teachers who showed a preference for flexible learning were found to be more successful in completing the program. The effects of PYP were measured by Objective Three: to determine the association between amount of PYP completed and factors related to the teaching of HIV lessons. Three of the six factors considered by this objective returned a significant association with program dose. Teacher perceived access to HIV education resources was found to be positively related to the dose of materials a participant completed. / Participants who completed a high dose of the program considered HIV resources to be relatively easier to access than participants completing a low dose. Teachers who completed a high dose of PYP reported being more comfortable to teach HIV lessons than teachers completing a mid dose. In addition, intervention group teachers showed a significant change in comfort with their ability to teach HIV lessons and specified HIV topics to Years 8, 9, and 10 classes and intervention group teachers of Year 8 students thought the HIV topics were less important for this level of students. The final variable to show a significant change over time when dose of the program was considered was teacher sexual conservativeness. Both high and mid dose participants reported being less sexually conservative than low or no dose participants from pre to midtest. The context of the teachers participating in the PYP study was investigated through Objective Four: to examine the context in which intervention and Comparison group teachers were operating for this study. Two factors were found to be associated with gender, six with school location and eleven with level of teaching. These associations provided important contextual information for interpreting the findings of the study. Objective Five evaluated the process of teacher recruitment to PYP, satisfaction with the flexible learning methodology, satisfaction with the PYP materials and completion of PYP. The recruitment strategies implemented for PYP were effective in having teachers from government and independent schools in WA recruited to PYP. / However, more than 90% of the intervention group were from government schools. Schools encouraged more than one teacher from a school to enrol, with nine primary schools, four district high schools, one community high school, one secondary college, four senior high schools and one combined independent primary and high school enrolling more than one teacher in the program. The flexible learning methodology was reported to be suitable for the needs of teachers who enrolled in PYP, as they felt comfortable with the learning methodology and appreciated the opportunity to choose when and where they completed the program. The opportunity for face-to-face contact, however, was still preferred by some teachers. The materials within the program were considered to be appropriate and useful. The writing style and activities were well received and the efforts of the tutors were welcomed by the majority of the intervention group. One third of teachers who enrolled in PYP completed at least some of the materials, but less than 10% completed the entire program. The most frequent suggestions made for increasing program completion rates were to set dates for completion of the program modules and to allow time release to complete the program. At baseline, this research showed that teachers considered it important for their students to have access to HIV education, but many of these teachers did not feel comfortable providing HIV education for their students. / As positive effects were observed in the PYP program of impact of program dose on factors affecting the implementation of HIV education, it would appear that flexible learning professional development was a suitable alternative to face-to-face professional development. Teachers' acceptance of flexible learning professional development as an alternate methodology, however, appears to be in its infancy and will require more empirical research. Future research, study design improvements and intervention design improvements can be informed by the following recommendations. Future research Recommendation 1: There be more rigorous investigation of flexible learning as a methodology for provision of professional development for teachers of health education. Recommendation 2: The status of claiming credit for professional development toward postgraduate qualifications for teachers continue to be investigated. Recommendation 3 : Further research be undertaken to evaluate available technologies and their acceptance by teachers as a delivery method for flexible learning professional development. Study design improvements Recommendation 4: design limitations of the PYP study. Future research be designed to overcome the study Intervention design improvements Recommendation 5: The findings of the PYP study and suggestions made by PYP participants be used to improve future health education professional development programs.

Einleitung/ Zielstellung Zusätzlich zur Gammastrahlung emittiert 99mTc ca. 5 niederenergetische Auger-Elektronen mit Reichweiten von wenigen Nanometern im Gewebe. Diese haben für die nuklear-medizinische Diagnostik keine Bedeutung. Es wird jedoch über eine therapeutische Nutzung diskutiert, wofür eine Anreicherung der Auger-Elektronen-Emitter in einem strahlensensitiven Zellkompartiment erforderlich ist. Ziel der Arbeit war es, verschiedene [99mTc]Tc-Radiopharmaka hinsichtlich ihres Uptakeverhaltens, der subzellulärer Verteilung und des Retentionsverhaltens in vitro zu untersuchen, sowie die mutmaßlich durch den Auger-Effekt hervorgerufene Radiotoxizität der [99mTc]Tc-markierten Radiopharmaka zu vergleichen und die gewonnenen Ergebnisse in Hinblick auf potentielle extranukleäre strahlensensitive Targets zu interpretieren. Material und Methode Durchgeführt wurden die Versuche im ersten Abschnitt der Arbeit an Natrium-Iodid-Symporter (NIS)-positiven FRTL-5-Schilddrüsenzellen. Von [99mTc] Pertechnetat ([99mTc]TcO4-), [99mTc]TcO4- nach Vorinkubation von Perchlorat ([99mTc]TcO4-/ ClO4-), [99mTc]Tc-Hexakis-2-Methoxyisobutylisonitril ([99mTc]Tc-MIBI), [99mTc]Tc-Hexamethyl-Propylenaminoxim ([99mTc]Tc-HMPAO) und [99mTc]TcO4- nach Vorinkubation von Zinn-Pyrophosphat (Sn- PYP/ [99mTc]TcO4-) wurden die intrazelluläre Radio¬nuklid¬aufnahme und die subzelluläre Verteilung untersucht. Basierend auf den Ergebnissen dieser Versuche wurde die mittlere absorbierte Zellkerndosis kalkuliert. Zur Beurteilung der strahlenbiologischen Wirkung wurde das klonogene Zellüberleben mit der Anzahl residualer gH2AX-Foci (DNA-Schaden) verglichen und die Wirkung der [99mTc]Tc Tracer auf den Zellzyklus von FRTL-5-Zellen untersucht. Im zweiten Abschnitt der Arbeit wurde an EGFR-positiven A431-Zellen die radiotoxische Wirkung in Abhängigkeit von der intra¬zellulären Lokalisation von [99mTc]Alexa(488)-C225-Cyclooctin-Dpa-Tc(CO)3 ([99mTc]Tc-C225), [99mTc]Tc-HMPAO und [99mTc]TcO4- auf das klonogene Zellüberleben untersucht. Ergebnisse und Diskussion Aufgrund verschiedener Uptakemechanismen zeigte jedes der untersuchten [99mTc]Tc-Radiopharmaka Unterschiede im zeitlichen Verlauf der Uptakekinetik. Durch Blockierung des NIS durch ClO4- konnte eine intrazelluläre Aufnahme von [99mTc]TcO4- verhindert werden, wogegen durch Vorinkubation mit Sn-PYP die zelluläre Aufnahme von [99mTc]TcO4- um das 22-fache gesteigert wurde. [99mTc]Tc-MIBI und [99mTc]Tc-HMPAO wurden aufgrund ihrer lipophilen Eigenschaften unabhängig vom NIS passiv in die Zelle transportiert. Die Untersuchung der intrazellulären Verteilung ergab für [99mTc]Tc-HMPAO und Sn-PYP/ [99mTc]TcO4- eine vergleichbar hohe Anreicherung in der Membran/Organellen-Fraktion sowie in der Zellkernfraktion. Von [99mTc]TcO4- und [99mTc]Tc-MIBI wurde die Hauptaktivität in der Zytosol-Fraktion und nur geringe Anteile in der Membran/Organellen-Fraktion sowie in der Zellkernfraktion nachgewiesen. In guter Übereinstimmung zur subzellulären Verteilung zeigten Sn-PYP/ [99mTc]TcO4- und [99mTc]Tc-HMPAO eine fast vollständige, hingegen [99mTc]Tc-MIBI und [99mTc]TcO4- nur eine geringe Retention. Aufgrund der genannten Unterschiede wurde bei gleicher inkubierter Aktivitätskonzentration folgende Reihenfolge der resultierenden Zellkerndosis ermittelt: [99mTc]TcO4- < [99mTc]Tc-MIBI < [99mTc]Tc-HMPAO < Sn-PYP/ [99mTc]TcO4-. [99mTc]TcO4- und [99mTc]Tc-HMPAO zeigten eine ähnliche Wirkung auf das klonogene Zellüberleben und auf den Zellzyklus. Jedoch bewirken sie eine wesentlich stärkere Reduzierung des Überlebens und einen stärkeren G2-Arrest als [99mTc]Tc-MIBI und Sn-PYP/ [99mTc]TcO4-, wobei [99mTc]Tc-MIBI bei allen drei untersuchten biologischen Endpunkten die geringste Wirkung zeigte. Bei einer vergleichbaren Reduktion des Zellüberlebens von [99mTc]TcO4- und [99mTc]Tc-HMPAO induzierte [99mTc]Tc-HMPAO lediglich die Hälfte der gH2AX-Foci im Vergleich zu [99mTc]TcO4-. Die trotz geringerem DNA-Schaden vergleichbare radiotoxische Wirkung zeigte, dass das klonogene Zellüberleben nicht allein vom DNA-Schaden abhängt. Daraus folgt, dass es außer der Kern-DNA noch weitere strahlensensitive Kompartimente gibt, die durch [99mTc]Tc-HMPAO stärker geschädigt wurden als von den anderen untersuchten [99mTc]Tc Tracern. Ein mögliches extranukleäres strahlensensitives Target ist die Zellmembran, so dass im zweiten Teil der Arbeit zur Überprüfung der Radiosensitivität der Zellmembran die radiotoxische Wirkung von [99mTc]Tc-C225 an EGFR-positiven A431-Zellen untersucht wurde. [99mTc]Tc-C225 wurde über den EGFR und [99mTc]Tc-HMPAO aufgrund seiner Lipophilie durch Diffusion intrazellulär aufgenommen. [99mTc]TcO4- dagegen zeigte keine intrazelluläre Aufnahme in die NIS-negativen Zellen und wurde als Referenz für eine extrazelluläre Bestrahlung verwendet. [99mTc]Tc-C225 wies nach einstündiger Inkubation eine Membranbindung von lediglich 10 % auf, die im Laufe von 24 h auf 1,9 % absank. Dies zeigte, dass [99mTc]Tc-C225 rasch in den A431-Zellen internalisiert wurde und dass nur bei sehr kurzen Inkubationszeiten von einer spezifischen Zellmembranmarkierung gesprochen werden kann. [99mTc]Tc-HMPAO ging keine Bindung an die Zellmembran ein. Weiterhin wurde bei der Inkubation steigender Aktivitäts- und Antikörperkonzentrationen von [99mTc]Tc C225 eine Sättigung des EGFR beobachtet, woraus eine wesentlich geringere Zellkerndosis als bei Inkubation von [99mTc]Tc-HMPAO resultierte. Im Vergleich des klonogenen Zellüberlebens zeigten [99mTc]Tc-C225 und [99mTc]Tc-HMPAO bei gleicher Zellkerndosis keine Unterschiede in der radiobiologischen Wirkung. Somit konnte lediglich eine Verstärkung der radiotoxischen Effekte von [99mTc]Tc-C225 an A431-Zellen im Vergleich zur ausschließlich extrazellulären Verteilung von [99mTc]TcO4- gezeigt werden. Schlussfolgerung Die Untersuchung der radiotoxischen Wirkung von [99mTc]Tc-C225 ermöglichte bei den angewendeten Versuchsbedingungen keine Rückschlüsse auf die Strahlensensitivität der Zellmembran. Weiterführende Arbeiten zur Entwicklung eines 99mTc-markierbaren spezifischen Membranmarkers wären notwendig, um klären zu können, ob die Zellmembran ein ähnlich strahlensensitives Target wie die nukleäre DNA ist. Dosimetrische Betrachtungen an den als Modellsystemen dienenden FRTL-5- und A431-Zellen deuten darauf hin, dass aufgrund ungenügender Anreicherung eine therapeutische Wirkung der Auger-Elektronen im Tumorgewebe eher unrealistisch ist. Damit sollte aus gegenwärtiger Sicht die klinische Anwendung von 99mTc auf den diagnostischen Einsatz beschränkt bleiben. Jedoch könnte 99mTc als Auger-Elektronen-Emitter bei spezifischer Anreicherung in definierten Zellkompartimenten als Nano-Tool zur Erforschung der Strahlensensitivität einzelner Zellbestandteile eingesetzt werden. / Introduction In addition to gamma radiation, 99mTc emits approximately 5 low energy Auger and internal conversion electrons per decay, resulting in high ionization density proximal to the radionuclide’s decay position. Low-energy Auger electrons with path lengths of only nanometers cannot be utilized for diagnostic procedures; however, they have frequently been discussed for therapeutic applications. To achieve a radiobiological effect, an intracellular accumulation and distribution in relevant cell compartments of the Auger electron emitter is required. Aim The aim of the thesis was the comparison of different [99mTc]Tc-labeled compounds concerning their intracellular uptake, subcellular distribution and retention in vitro. Furthermore the radiotoxicity caused by the Auger effect has to be investigated. Material and Methods The intracellular radionuclide uptake, subcellular distribution (ProteoExtract®-Kit) and retention of [99mTc] pertechnetate ([99mTc]TcO4-), [99mTc]TcO4- after pre-incubation of perchlorate ([99mTc]TcO4-/ClO4-), [99mTc]TcO4- after pre-incubation of stannous pyrophosphate ([99mTc]TcO4-/Sn-PYP), [99mTc]Tc-hexamethyl-propylene-aminoxime ([99mTc]Tc-HMPAO) and [99mTc]Tc-hexakis-2-methoxyisobutylisonitrile ([99mTc]Tc-MIBI) were quantified in sodium-iodide symporter (NIS)-positive rat thyroid FRTL-5 cells. Basing on these results the mean absorbed nucleus dose was calculated. Radiotoxicity was investigated using phosphorylated histone H2AX (gH2AX foci), clonogenic cell survival and cell cycle analyzes. Additionally the radiotoxicity of [99mTc]Alexa(488)-C225-Cyclooctin-Dpa-Tc(CO)3 ([99mTc]Tc-C225) was compared with the one of [99mTc]TcO4- and [99mTc]Tc -HMPAO depending on the subcellular distribution in EGFR-positive A431 cells. Results and Discussion For the analyzed [99mTc]Tc-labeled compounds we detected differences in the time courses of the uptake kinetics caused by different uptake mechanisms into the FRTL-5 cells. The radionuclide uptake of [99mTc]TcO4- was blocked in the presence of perchlorate and increased by a factor of approximately 22 after pre-incubation of Sn-PYP. The lipophilic complexes [99mTc]Tc-MIBI and [99mTc]Tc-HMPAO crossed the cell membrane through passive transport via diffusion. The compartmental analysis indicated that [99mTc]Tc-HMPAO and [99mTc]TcO4-/Sn-PYP revealed a comparable high uptake in the nucleus and in the membrane/organelle fraction. [99mTc]TcO4- and [99mTc]Tc-MIBI were preferentially distributed in the cytosol, with lower amounts of the accumulated activity in both the membranes/organelles and the nucleus compared with the other compounds. In good agreement with the subcellular distribution [99mTc]Tc-HMPAO, [99mTc]TcO4-/Sn-PYP showed a nearly complete retention and [99mTc]TcO4-, [99mTc]Tc-MIBI a low retention. Due to the differences mentioned above the following sequence of the calculated mean nucleus dose for identical activity concentrations was determined: [99mTc]TcO4- < [99mTc]Tc-MIBI < [99mTc]Tc-HMPAO < Sn PYP/ [99mTc]TcO4-. [99mTc]TcO4- and [99mTc]Tc-HMPAO caused a similar reduction of the cell survival and a dose dependent G2-arrest. [99mTc]Tc-MIBI and Sn-PYP/ [99mTc]TcO4- are both less radiotoxic in terms of the estimated nucleus dose compared with [99mTc]TcO4- and [99mTc]Tc-HMPAO. Despite the similar effect on the cell survival [99mTc]Tc-HMPAO induced only half of the residual gH2AX foci than [99mTc]TcO4-. These findings reveal that clonogenic cellular survival is not solely determined by the DNA-DSB response, which may suggest the involvement of extra-nuclear radiosensitive targets in cell inactivation. A possible extra-nuclear radiosensitive target is the cell membrane. That’s why the aim of the second part of the thesis is the investigation of the radiosensitivity of the cell membrane. Therefore the radiotoxic influence of [99mTc]Tc-C225 was analyzed at EGFR-positive A431 cells. [99mTc]Tc-C225 was taken up over the EGFR and the lipophilic [99mTc]Tc-HMPAO was transported via diffusion over the cell membrane. In contrast, [99mTc]TcO4- did not show any intracellular uptake into the NIS-negative cells and therefore was used as extracellular reference. An incubation of [99mTc]Tc-C225 for one hour resulted to a membrane binding of only 10 %, which was reduced to 1.9 % after 24 hours. This demonstrated a fast internalization into A431-cell. Therefore only in the case of a very short incubation time [99mTc]Tc-C225 leads to a specific targeting of the cell membrane. [99mTc]Tc-HMPAO did not bind to the cell membrane. Furthermore the incubation of increasing concentrations of activity and antibody resulted in a saturation of the EGFR, leading to a significant lower nucleus dose in comparison to the incubation of [99mTc]Tc-HMPAO. Concerning the clonogenic cell survival no differences in the radiotoxicity of [99mTc]Tc-C225 and [99mTc]Tc-HMPAO were observed for equal nucleus dose. Thus only an amplification of the radiotoxic effects of [99mTc]Tc-C225 in comparison to the extracellular distribution in A431 cells of 99mTc-pertechnetate was observed. Conclusion The investigation of the radiotoxic effect of [99mTc]Tc-C225 did not allow any conclusions about the radiosensitivity of the cell membrane under the given experimental conditions. For clarifying if the radiosensitivity of the cell membrane is comparable to the one of the nucleus DNA further experiments for the development of a [99mTc]Tc-labeled specific target for the cell membrane are necessary. On the basis of the dosimetric considerations of the FRTL-5 cells and A431 cells used as model systems it can be concluded that because of an insufficient accumulation a therapeutic radiotoxic effect of the Auger electrons is not realistic. Therefore the clinical use of 99mTc should be limited to the diagnostics. Nevertheless specific accumulated Auger electrons of 99mTc could be applied in the field of investigation as nano-tools for the subcellular analysis of radiotoxicity.

Halorhodospira halophila photoactive yellow protein (PYP) is a promising candidate to act as a photoswitching domain in engineered proteins due to the structural changes that occur during its photocycle. Absorption of a photon of wavelength 446 nm triggers trans to cis isomerization of its 4-hydroxycinnamic acid chromophore leading to large structural perturbations in the protein, particularly in the N-terminus. In the dark, a slower cis to trans reisomerization of the chromophore restores the protein’s native fold. The fusion of proteins to PYP’s N-terminus may therefore enable photomodulation of the activity of the attached protein. To test this hypothesis, this thesis descibes genetically encoded photoswitchable DNA-binding proteins that were developed by fusing the prototypical leucine-zipper type DNA-binding protein GCN4 bZIP to the N-terminus of PYP. Five different fusion constructs of full length or truncated GCN4 bZIP and full length PYP as well as fusion constructs of full length GCN4 bZIP and N-terminally truncated PYP mutants were designed in a structure-based approach to determine if the dimerization and DNA binding activities could be controlled by the PYP photocycle. Extensive biophysical characterization of the fusion constructs in the dark and under blue light irradiation using electronic absorption, circular dichroism and fluorescence spectroscopic techniques were performed. As all the fusion proteins could complete photocycles, the DNA binding abilities of the dark and light-adapted states of the proteins were characterized using spectroscopic techniques as well as by the electrophoretic mobility shift assay. All the fusion constructs maintained DNA-binding abilities, however they each differed in their affinities and the extent to which they were activated by blue light irradiation. The reasons for these differences in DNA-binding abilities and photoactivation are explored. Using the results from the characterization of these constructs, proposals are also made to develop more robust genetically encoded photoactivatable DNA-binding proteins of the same type.

Halorhodospira halophila photoactive yellow protein (PYP) is a promising candidate to act as a photoswitching domain in engineered proteins due to the structural changes that occur during its photocycle. Absorption of a photon of wavelength 446 nm triggers trans to cis isomerization of its 4-hydroxycinnamic acid chromophore leading to large structural perturbations in the protein, particularly in the N-terminus. In the dark, a slower cis to trans reisomerization of the chromophore restores the protein’s native fold. The fusion of proteins to PYP’s N-terminus may therefore enable photomodulation of the activity of the attached protein. To test this hypothesis, this thesis descibes genetically encoded photoswitchable DNA-binding proteins that were developed by fusing the prototypical leucine-zipper type DNA-binding protein GCN4 bZIP to the N-terminus of PYP. Five different fusion constructs of full length or truncated GCN4 bZIP and full length PYP as well as fusion constructs of full length GCN4 bZIP and N-terminally truncated PYP mutants were designed in a structure-based approach to determine if the dimerization and DNA binding activities could be controlled by the PYP photocycle. Extensive biophysical characterization of the fusion constructs in the dark and under blue light irradiation using electronic absorption, circular dichroism and fluorescence spectroscopic techniques were performed. As all the fusion proteins could complete photocycles, the DNA binding abilities of the dark and light-adapted states of the proteins were characterized using spectroscopic techniques as well as by the electrophoretic mobility shift assay. All the fusion constructs maintained DNA-binding abilities, however they each differed in their affinities and the extent to which they were activated by blue light irradiation. The reasons for these differences in DNA-binding abilities and photoactivation are explored. Using the results from the characterization of these constructs, proposals are also made to develop more robust genetically encoded photoactivatable DNA-binding proteins of the same type.

<p>The purpose of this study is to determine what alternative forms of assessments are being practiced in a public school with an international programme and to explore the teachers` attitudes towards the use of alternative assessment procedures. Various assessment models and strategies have been investigated and discussed, as pre¬senting a part of the educational practice in the primary classrooms that engage the International Baccalaureate/ Primary Years Programme at the elementary school level (age range 6-11) in the years 0-5.</p><p>While defining my research problem, I have started from hypothesis that practicing of alternative assessment has an important positive role in the international schools supporting, promoting and improving student learning.</p><p>International schools are facing both challenging and complexity of assessment pro¬cess while striving to apply both national and international programs` recom¬menda¬tions concerning the testing.</p><p>Alternative forms of assessments are being used in conjunction with other forms of assessment, such as standardised tests, in order to assess both student perfor¬mance and the intentions of the International Baccalaureate/ Primary Years Pro¬gramme.</p><p>This paper investigates what alternative assessment practices (portfolio, perfor¬mance assessment, Exhibition, self-assessment) are being applied and used in the same school where the standardised tests are also being applied and used (text book tests, teacher-made tests, local and national test).</p><p>The hypothesis was supported and the study findings suggest that various types of assessments are needed to be utilized in order to fairly evaluate students` needs as well as that alternative assessment has an important positive role meeting individual student’s needs supporting a process of learning.</p>

The purpose of this study is to determine what alternative forms of assessments are being practiced in a public school with an international programme and to explore the teachers` attitudes towards the use of alternative assessment procedures. Various assessment models and strategies have been investigated and discussed, as pre¬senting a part of the educational practice in the primary classrooms that engage the International Baccalaureate/ Primary Years Programme at the elementary school level (age range 6-11) in the years 0-5. While defining my research problem, I have started from hypothesis that practicing of alternative assessment has an important positive role in the international schools supporting, promoting and improving student learning. International schools are facing both challenging and complexity of assessment pro¬cess while striving to apply both national and international programs` recom¬menda¬tions concerning the testing. Alternative forms of assessments are being used in conjunction with other forms of assessment, such as standardised tests, in order to assess both student perfor¬mance and the intentions of the International Baccalaureate/ Primary Years Pro¬gramme. This paper investigates what alternative assessment practices (portfolio, perfor¬mance assessment, Exhibition, self-assessment) are being applied and used in the same school where the standardised tests are also being applied and used (text book tests, teacher-made tests, local and national test). The hypothesis was supported and the study findings suggest that various types of assessments are needed to be utilized in order to fairly evaluate students` needs as well as that alternative assessment has an important positive role meeting individual student’s needs supporting a process of learning.

In mammals, freshly ejaculated spermatozoa do not possess the ability to fertilize a mature oocyte. They acquire fertilization competence upon residing for a period of time in the female reproductive tract. The physiological changes that bring about these time-dependent changes in motility pattern and acquisition of fertilizing ability of spermatozoa are collectively referred to as capacitation, culminating in sperm hyperactivation. Capacitation-associated increase in sperm protein tyrosine phosphorylation (PYP), exhibited by mammalian sperm, is one of the major downstream events, regulating hyperactivated motility. However, it is still unclear which are the tyrosine kinases and phosphatases involved in modulating the capacitation-associated increase in global PYP. In order to determine this, our laboratory earlier showed the role of PYP in hamster sperm capacitation using a specific EGFR protein tyrosine kinase (PTK) inhibitor, tyrphostin A47 (TP-47). Interestingly, inhibition of capacitation by 0.5 mM TP-47 was associated with induction of a slow circular motility pattern, accompanied by inhibition of PYP of certain proteins (Mr. 45,000-52,000), localized to the principle piece of the sperm flagellum. Two such proteins, hypo-tyrosine phosphorylated, were found to be tektin-2 and ODF-2, using 2D-PAGE followed by MS/MS analysis. Interestingly, a global phosphoproteome analysis of human spermatozoa showed that PYP changes are associated with capacitation and asthenospermic condition in infertile men is attributed to the failure of capacitation-associated increase in PYP. Such individuals exhibited impaired sperm motility. There is a need to understand the exact mechanism of phosphorylation of sperm flagellar proteins, which is necessary to assess sperm’s ability to fertilize the mature oocyte. Therefore, the focus of the present work was to elucidate the role of receptor tyrosine kinases (RTKs) and the non-receptor tyrosine kinases (NRTKs) in mammalian (hamster) sperm capacitation. Recent studies have shown that apart from EGFR other RTKs like IGF1R, FGFR, VEGFR, MuSK, TrkA are expressed in mammalian spermatozoa and actively involved in sperm capacitation. However, there is very little information available in the context of sperm capacitation and associated PYP. Therefore, attempts were made to understand the role of various RTKs (IGF1R, FGFR and VEGFR) in hamster sperm capacitation and associated PYP. Initially, the role of IGF1R tyrosine kinase during sperm capacitation was studied. Immunolocalization of IGF1R in spermatozoa showed a strong signal in the sperm acrosome and the principal piece of the sperm flagellum. Inhibition of IGF1R kinase with an IGF1R-specific inhibitor TP-1-O-Me-AG538 (TP-538) showed inhibitory effect on sperm capacitation and the associated hyperactivation. But, inhibitors of FGFR and VEGFR tyrosine kinases did not show such an effect. Interestingly, inhibition of IGF1R by TP538 was associated with inhibition of PYP of certain proteins (Mr. 45,000-120,000), localized to head, mid piece and principle piece regions of the sperm flagellum. Phosphoproteomic analysis using 2D-PAGE-western blot with anti-phosphotyrosine antibodies identified 17 differentially phosphorylated protein spots. Out of the 17 spots, 12 were identified by MALDI-MS/MS analysis. The proteins identified to be differentially phosphorylated, upon inhibition of IGF1R, were PDHE1, ODF-2, Tubulin β 2C chain, PDHE2 and ATP synthase β subunit. The RTKs being present in the membrane level may not be directly involved in the phopshorylation of downstream target proteins associated with the mitochondrial membrane, sperm axonemal structures and outer dense fibers. Therefore, the RTKs may interact directly or indirectly with the downstream NRTKs, which may be involved in the phosphorylation of target sperm proteins. Till date, six different families of NRTKs are shown to be expressed in mammalian spermatozoa. The major family of NRTKs involved in sperm function is the Src family of kinases. However, there is very little information available in the context of sperm capacitation and the associated PYP. Therefore, studies were carried out to understand the role of Src family of NRTKs in sperm capacitation and associated PYP. Presence of active Src signaling was observed by the immunolocalization of activated Src (pY416) in the acrosome, mid piece and the principal piece regions of the sperm flagellum. Inhibition of Src family of kinase with a specific Src family kinase inhibitor PP2, showed inhibition of sperm capacitation and the associated hyperactivation. Inhibition of Src family of kinases with PP2 was associated with decrease in PYP of several proteins (Mr. 45,000-120,000), localized mainly to the mid piece region, followed by the principle piece region of the sperm flagellum. Phosphoproteomic analysis using 2D-PAGE-western blot with anti-phosphotyrosine antibodies identified 38 differentially phosphorylated protein spots. Out of the 38 spots, 16 were identified by MALDIMS/MS analysis and these corresponded to seven proteins which included PDHE1, ODF-2, Tubulin β 2C chain, Tektin-2, GAPDS, PDHE2 and ATP synthase β subunit. Additionally, the biochemical and molecular characteristics of the identified proteins were also studied. Bioinformatic analysis predicted the presence of phosphorylation motifs for several kinases and interestingly, all the proteins identified had a Src kinase motif. Comparing the current observations and the previous work in the laboratory, two proteins ODF-2 and Tektin-2 were found to be regulated by EGFR, IGF1R and Src family of kinases. Therefore, characterization of the capacitation-associated tyrosine phoposphorylated proteins ODF-2 and Tektin-2 was performed. By employing PCR and Northern blotting techniques, the presence of the transcripts of both the proteins was shown. Additionally, the ontogeny of expression of ODF2 and Tektin-2 in hamster testis development was studied and the results indicated that the expression of both the proteins started from week 3 onwards till week 8. To confirm the meiotic stage-associated expression of ODF-2 and Tektin-2, germ cells were sorted based on their DNA content. ODF-2 and Tektin-2 transcripts were first expressed in the meiotic germ cells (pachytene spermatocytes) and their expression was upregulated in the post-meiotic germ cells (round spermatids). Sequential extraction of sperm proteins showed that, Tektin-2 was majorly extracted out in the Triton X-100 and DTT fraction, whereas, ODF-2 was maximally extracted in the presence of urea and DTT. In conclusion, these observations indicate that IGF1R and Src family of tyrosine kinases are critical for mammalian sperm capacitation and associated global PYP. Inhibition of sperm capacitation was associated with hypo-tyrosine-phospohorylation of certain proteins associated with mitochondrial membrane, axonemal structures and outer dense fibers of the sperm flagellum. Future work can be directed towards understanding the role of other RTKs and NRTKs involved in sperm capacitation and the molecular characterization of hypophosphorylated proteins critical for sperm function and its fertilization competence.