Calcium transporters regulate calcium fluxes within cells. Plants, like all organisms, contain channels, pumps and exchangers to carefully modulate intracellular calcium levels. Numerous Arabidopsis proteins have been characterized which can transport calcium. However, there are numerous genes whose products have not been fully characterized. One method I used to infer function was to analyze various promoter lengths of 17 putative cation transporters fused to reporter and to observe changes in the reporter expression in response to various stimuli. Using a more in depth approach I set out to characterize the function of AtCCX3. Here I show Arabidopsis AtCCX3 can suppress yeast mutants defective in vacuolar Na+ and K+ transport. AtCCX3-expressing yeast cells conferred Mn2+ sensitivity when highly expressed. Functional epitope tagged AtCCX3 fusion proteins were localized to endomembranes in plants and yeast. Expression of AtCCX3 increased in plants treated with NaCl, KCl and MnCl2 and caused increased Na+ accumulation and increased K+ transport. Ectopic expression of AtCCX3 in tobacco produced lesions in the leaves, stunted growth, resulted in the accumulation of higher levels of numerous cations and increased protein oxidation preceded alterations in leaf growth. These characteristics define AtCCX3 as an endomembrane localized transporter with biochemical properties distinct from previously characterized plant transporters. In the remaining studies I used the mouse model to determine how molecular changes to plants can improve the nutritional quality of the food. Previously, the cod5 M. truncatula mutant was identified which contains identical calcium concentrations to wild-type, but contains no oxalate crystals. Mice fed intrinsically labeled cod5 plants had 22.87% (p<0.001) calcium absorption compared to wild type plants. In a second study, using mice I examined if increased expression of a calcium transporter which increases calcium concentration 2-fold, alters bioavailable calcium. In mice feeding regimes (n = 120), I measured 45Ca-incorporation into bones, and determined that mice required twice the serving size of control carrots to obtain the calcium found in sCAX1-carrots. Together, these two studies demonstrate how the potential utility of removing calcium absorption inhibitors and fortifying vegetables can improve calcium bioavailability.

Calcium transporters regulate calcium fluxes within cells. Plants, like all organisms, contain channels, pumps and exchangers to carefully modulate intracellular calcium levels. Numerous Arabidopsis proteins have been characterized which can transport calcium. However, there are numerous genes whose products have not been fully characterized. One method I used to infer function was to analyze various promoter lengths of 17 putative cation transporters fused to reporter and to observe changes in the reporter expression in response to various stimuli. Using a more in depth approach I set out to characterize the function of AtCCX3. Here I show Arabidopsis AtCCX3 can suppress yeast mutants defective in vacuolar Na+ and K+ transport. AtCCX3-expressing yeast cells conferred Mn2+ sensitivity when highly expressed. Functional epitope tagged AtCCX3 fusion proteins were localized to endomembranes in plants and yeast. Expression of AtCCX3 increased in plants treated with NaCl, KCl and MnCl2 and caused increased Na+ accumulation and increased K+ transport. Ectopic expression of AtCCX3 in tobacco produced lesions in the leaves, stunted growth, resulted in the accumulation of higher levels of numerous cations and increased protein oxidation preceded alterations in leaf growth. These characteristics define AtCCX3 as an endomembrane localized transporter with biochemical properties distinct from previously characterized plant transporters. In the remaining studies I used the mouse model to determine how molecular changes to plants can improve the nutritional quality of the food. Previously, the cod5 M. truncatula mutant was identified which contains identical calcium concentrations to wild-type, but contains no oxalate crystals. Mice fed intrinsically labeled cod5 plants had 22.87% (p<0.001) calcium absorption compared to wild type plants. In a second study, using mice I examined if increased expression of a calcium transporter which increases calcium concentration 2-fold, alters bioavailable calcium. In mice feeding regimes (n = 120), I measured 45Ca-incorporation into bones, and determined that mice required twice the serving size of control carrots to obtain the calcium found in sCAX1-carrots. Together, these two studies demonstrate how the potential utility of removing calcium absorption inhibitors and fortifying vegetables can improve calcium bioavailability.

Osteoporosis is a disease of the bone and skeletal system which weakens bone structure and results in fractures. The disease has caused a heavy economic burden in the U.S., especially among Americans over the age of 50. According to The Surgeon General's report on bone health in 2004 (U.S. Department of Health and Human Service (USDHHS), 2004), an estimated 10 million Americans over age 50 have osteoporosis, and another 34 million have osteopenia and are at risk of osteoporosis. The projected cost directly associated with osteoporosis is $34.0 billion in 2005 and will rise to more than $41.4 billion in 2025 (Vanness & Tosteson, 2005). Osteoporosis also results in serious and often devastating health problems for affected individuals (Riggs & Melton, 1995) because of the fractures it causes. Riggs and Melton (1995) estimated that each year 1.5 million people suffer an osteoporosis-related fracture. Unlike genetic risk factors that can be nearly impossible to modify, living on a calcium-rich diet is a lifestyle choice. Effective intervention programs can be useful tools to educate people to adopt a calcium-rich diet. Studies have shown that a calcium-rich diet can increase bone mineral density and intervention programs can facilitate the process of the lifestyle change (Wong, Lau, E.M., Lau, W.W., & Lynn, 2004; Lv & Brown, 2011). However, effective intervention programs against osteoporosis are limited for minorities, and specifically, for elderly Chinese women. The prevalence of osteoporosis in Chinese women is high but the knowledge level is low (Babbar et al., 2006; Lau, Woo, Leung, Swaminathan, & Leung, 1992), which provides an opportunity for intervention. A community-based intervention program was designed based on the modified health belief model (HBM). Its content was specifically tailored to the cultural background and the characteristics of the Chinese women living in the Greater Philadelphia area, based on the findings from previous studies. The intervention program was implemented and its effectiveness was evaluated in a 2-group quasi-experimental study. The study recruited eight (8) Chinese community organizations and assigned them to receive either the intervention program or the control program according to the timing of their agreement to participate and the balance of total participants recruited for each study group at the time. A total of 102 Chinese women were enrolled from the three (3) organizations that were assigned to the Intervention Group and 90 from the five (5) organizations that were assigned to the Control Group. Participants in the Intervention Group received a 30-mintue education session delivered in Chinese (Mandarin). The education focused on the Health Belief Model constructs in the context of osteoporosis intervention. It provided information about osteoporosis, including the functions of the bones, prevalence and risk factors. It highlighted participants' susceptibility to osteoporosis, the consequences and severity of the disease, and targeted messages to increase self-efficacy and decrease barriers. The contents were tailored to the study population. The osteoporosis education was delivered in plain language to suit the population's low educational level and used examples relevant to their cultural background. Participants in the Control Group watched a 30-minute video about liver functions and hepatitis B transmission and prevention. The study tested three (3) primary hypotheses that, compared with the Control Group, the Intervention Group at post-intervention would have 1) a higher knowledge level of osteoporosis, 2) a higher self-efficacy for adopting a calcium-rich diet, and 3) a higher intention level to consume more calcium-rich food items. The data were collected at three time points: baseline, post-intervention, and three months after the intervention. The scores of knowledge level and self-efficacy were analyzed by mixed linear regression models with adjustment of the baseline variables and accounted for the correlations among the participants from the same site. The intention stage was analyzed by a multinomial logistic regression model with adjustment of baseline intention stage and the baseline variables. The results for the study were consistent with all three primary hypotheses. The intervention program increased the knowledge level of osteoporosis and perceived self-efficacy in the Intervention Group. At post-intervention, the differences between the two study groups was 0.17 (95% CI: 0.02, 0.32; p-value&lt;0.037) for the knowledge scores, and 0.34 (95% CI: 0.12, 0.56; p-value&lt;0.001) for the self-efficacy scores. The results also suggested that the intervention program moved the participants in the Intervention Group to higher intention stages. The odds ratio (Intervention/Control Group) of being at a higher intention stage was 3.29 (95% CI: 1.23, 8.82; p-value=0.016). The study showed that the community-based and culturally tailored intervention program designed for the elderly Chinese women was effective. It increased the osteoporosis knowledge level and self-efficacy for adopting a calcium-rich diet and moved participants to a higher intention stage of consuming more calcium-rich food items. Additionally, the study showed that more than 50% of the participants had low bone mass. In conclusion, this intervention program reached a hard-to-reach population of elderly Chinese women and provided public health professionals a useful tool to work with. The high provalance of low bone mass in this population provided the public health agencies useful information to aid their decisions on resource allocation. / Public Health

Die Entdeckung der Kanalrhodopsine (ChRs) C1 und C2 aus der Grünalge Chlamydomonas reinhartii vor 10 Jahren brachte die neue Proteinfamilie der lichtgesteuerten Ionenkanäle hervor. Diese 7-Transmembranproteine verwenden all-trans Retinal, um Licht einer bestimmten Wellenlänge zu absorbieren. Bei Lichtabsorption isomerisiert das Retinal und das Protein öffnet daraufhin seine Pore, die es Ionen wie H+, Na+ und Ca2+ erlaubt, abhängig von ihrem elektrochemischen Gradienten, die Membran zu passieren. Insbesondere in der Neurophysiologie findet seit kurzem das C2 eine breite Anwendung, da seine Expression in Nervenzellen es erlaubt, mittels kurzer Lichtpulse die elektrische Aktivität dieser Zellen zu kontrollieren. In dieser Arbeit wurden zum ersten Mal die beiden neuen ChRs V1 und V2 aus der mehrzelligen Grünalge Volvox carteri elektrophysiologisch charakterisiert. Hierbei zeigte sich, dass V2, ähnlich wie C2, im blauen Spektralbereich absorbiert und auch, dass sich die kinetischen Eigenschaften stark ähneln. Dagegen besitzt V1 eine um 70 nm längerwellig verschobene Absorption auf 535 nm bei einer ~ 10 x langsameren Abklingkinetik als C2. Durch die schlechte Membranintegration von V1 in eukaryotischen Zellen ist der Photostrom gering und die Anwendungen in Neuronen stark eingeschränkt. Um die Membranintegration zu verbessern, wurden einzelne Helices von V1 gegen die entsprechenden Helices der anderen ChRs ausgetauscht. Hierbei wurde eine Chimäre C1V1-25, die aus den ersten 2 Helices von C1 und den restlichen 5 Helices von V1 besteht, entwickelt. Die Chimäre besitzt weiterhin eine rotverschobene Absorption bei 539 nm, einen 4 x höheren Photostrom als V1 und der doppelt so hoch ist wie der von C2. / The discovery of the channelrhodopsins (ChRs), C1 and C2 from the green alga Chlamydomonas reinhardtii 10 years ago, created the new protein family of light-gated ion channels. Those 7-Transmembranproteins are utilizing all-trans retinal to absorb light at specific wavelength. Upon light absorption the retinal isomerizes which leads to opening of a protein pore allowing ions such as H+, Na+ and Ca2+ to pass the membrane depending on their electrochemical gradient. In the last years C2 attracted a lot of attention in neuroscience since its expression in neurons allows to control their electrical properties with short light pulses. This work presents the electrophysiological characterization of two new ChRs V1 and V2 from the multicellular agla Volvox carteri for the first time . V2 absorbs light in the blue visible range like C2 and almost identical kinetic properties. In contrast, V1 exhibit a 70 nm redshifted absorption towards 535 nm and a ~ 10 x slower off-kinetic than C2. Since V1 displays only weak membrane targeting the resulting overall small photocurrent in eukaryotic cells significantly hampered its application in neuronal cells. To improve membrane targeting and to retain redshifted absorbance helices from V1 were exchanged with the corresponding helices from the other ChRs. A chimera called C1V1-25, which consists out of the first 2 helices from C1 and the last 5 helices from V1 showed a absorbance maximum at 539 nm and exhibit a 4 times higher photocurrent even beeing 2 times higher then the one of C2.