Breast cancer is one of the most dangerous forms of cancer worldwide. Continuous research is therefore essential in order to find realistic treatment options. 3D tumor spheroids have been proven to imitate the structural and physiological TME of in vivo tumors relatively more accurate than the more widely used 2D cell cultures. During this study, the aim was to find the optimal conditions for single spheroid formation, employing breast cancer cells and utilizing GrowDex. Different concentrations and cell densities were used, as well as differing methods (GrowDex addition prior or (days) after cell cluster formation). The liquid overlay technique was used to grow the spheroids, which were observed for a maximum of 14-16 days. Finally, an ATP viability assay was performed to determine whether the spheroids contained a proliferation zone, which corresponds to active cells. Single spheroids were obtained for all cell densities. The relatively high GrowDex concentrations responded well to a cell density of 2000 cells, while the relatively low GrowDex concentrations responded well to cell densities of 5000 and 10000 cells. Through the ATP viability assay the samples illustrated the presence of a proliferation zone, and thus active cells.

Because diagnostics have improved tremendously, nowadays breast cancer is more frequently detected at an early stage. Long-term prognosis is excellent and therefore the new treatment should focus on minimizing long-term side effects and treatment burden. This research proposes cancer treatment using nanoparticles (NPs) for a combination of heat therapy and brachytherapy. For the NP design, superparamagnetic iron-oxide nanoparticles (SPIONs) will be used. Because of their biocompatibility and good magnetic properties, they are suitable candidates for heating using an alternating magnetic field. To make the NPs applicable for brachytherapy, the NPs are doped with holmium (Ho), a lanthanide with high magnetic moment and radioisotope Ho-166. Furthermore, SPIONs and Ho change the relaxivity of water protons and therefore they can be used as contrast agents for magnetic resonance imaging. This makes it possible to image the NPs during the treatment and enables thermal planning and dose-rate calculations delivered to the cancer tissue to be performed. The incorporation of Ho inside the iron-oxide structure is expected to change the magnetic properties of the NPs. The success of using Ho as contrast agent and for brachytherapy purposes have been proven in several researches. However, the influence of Ho on the heating performance of these SPIONs is unknown. This research will characterize the magnetic properties of undoped and Ho-doped SPIONs and compare them, in order to examine the heating possibilities of Ho-doped NPs. For the synthesis of the NPs a spark discharge generator was used. Spark ablation is a synthesis method able to yield narrow size distribution of different metallic particles and alloys. However, since spark ablation is relatively new, the synthetic methodology itself had to be developed. Therefore, this research was divided into two parts: 1) the synthesis of NPs using spark ablation and 2) the characterization of Ho-doped iron NPs and the comparison with undoped SPIONs. Since the first part focused on the spark ablation synthesis, the influence of the generator settings on the primary particle size was investigated using DLS and TEM. The VS-Particle generator was able to produce 4 nm Fe-Fe and Ho-Fe NPs, a size that is smaller than expected. The generator settings did not influence the primary particle size and therefore, the settings of the generator were chosen to maximize the yield. The initial set-up had low yield and therefore, the influence of micro-bubbler, electrode configuration and bubbling column were examined and changed to improve the yield. A bronze sintered filter with pore size 0.4-20 ��, solid iron electrodes and 60 mL bubbling column volume appeared to be the most optimal. During the second part of this research, 4 nm Ho-doped iron oxide NPs and un-doped iron oxides were produced. They were characterized using TEM, DLS, XRD, ICP, SEM-EDX, Mössbauer spectroscopy, SQUID and NMR. As a result of low concentrations obtained, no good qualification of the produced particles could be obtained. All particles showed superparamagnetic behavior and influenced the relaxivity times of water protons. To what extend the relaxivity was changed upon doping remains unfortunately unclear, because the composition of the NPs is unknown. It can be concluded that the used set-up was not able to produce reproducible NPs and therefore no reliable characterization could be done on the Ho doped particles. Future research should focus on a more controllable synthesis method to be able to examine the possibilities of Ho for heating purposes.

Calcium is an important nutrient for both young and elderly people which can, among others, stimulate healthy bone growth and prevent osteoporosis. Therefore, nutrition with sufficient calcium is of great importance. The highest calcium nutrient density for all foods can be found in bovine milk, which contains on average 1.2 g Ca / L. To increase the amount of bioavailable calcium, milk can be fortified by the addition of different calcium salts. In this research the bioavailability and exchangeability of calcium in unfortified and fortified skimmed milk was studied by in vitro methods using equilibrium dialysis and ultracentrifugation to separate different fractions in milk. Radioactive 45Ca was used to follow the exchange behaviour of calcium between the three different fractions in milk: casein micelles, serum proteins, and soluble calcium. For unfortified milk it was found that 34.9 ± 2.2% of the calcium was present in the soluble phase, 9.1 ± 0.6% was bound to serum proteins, and 56 ± 1.6% was present in the casein micelles, which confirmed the results in literature. In addition, with the investigation of the exchange behaviour of 45Ca, it was confirmed that ~40% of calcium in casein micelles is hardly exchanged.To investigate the influence of calcium fortification on the calcium equilibrium in milk, six different calcium salts were selected to be synthesized: calcium chloride, calcium carbonate, calciumgluconate, tri-calcium di-citrate, calcium lactate, and tri-calcium phosphate. Of these, calcium chloride, calcium carbonate, calcium gluconate, and tri-calcium di-citrate were successfully synthesized. Further experiments were performed with calcium chloride and calcium carbonate, intrinsically labelled with 45Ca. From fractionation with ultracentrifugation, it was concluded that addition of calcium chloride led to an increase of calcium in the soluble phase, which might indicate an increase in bioavailability. Fortification of milk with 45Ca labelled calciumcarbonate did not lead to an exchange of 45Ca with the calcium present in milk, indicating that calcium carbonate is unlikely to make calcium in milk more bioavailable. In conclusion, a better understanding of the solubility and exchangeability of Ca in milk and the influence of milk fortification with calcium chloride and calcium carbonate is obtained by using 45Ca as a tracer.

Combining different treatment modalities has proven to improve patient outcome compared to applying single treatments. Consequently, mild hyperthermia (HT) and radiotherapy (RT) are combined to radiosensitize tumour cells by inhibiting DNA damage repair mechanisms. In this study, it was investigated if combining both treatment modalities shows an improved therapeutic effect on 3D tumour models made of FaDu cells. To this end, the spheroid size and cell viability were measured over time after treatment. It was also attempted to obtain further qualitative information of the effect on the spheroids using confocal microscopy. During experiments spheroids were formed of FaDu tumour cells, cells of a head-and-neck squamous cell carcinoma, using Matrigel®. These spheroids grew fast within the first 10 days of seeding up to a size of 550-600 𝜇m reliably. These spheroids were then exposed to both single HT and RT as well as combined HT and RT. Both single treatment modalities showed a decrease in cell growth and cell viability with increasing treatment dose. The thermal doses of 30 and 120 CEM43 and radiation doses of 2 and 6 Gy were chosen to be used in the combined treatment experiments. Combined treatment showed an improved effect on the cell growth and cell viability for most treatment groups, both for HT followed by RT and the other way around, with the determining factor seemingly being the thermal dose. The thermal dose of 120 CEM43 combined with 6 Gy of radiation dose showed the highest cell killing potential, with 9±2% of the cell viability remaining when using radiotherapy before hyperthermia and 18±3% when starting with HT before using RT. The most significant difference in effect due to the order in which RT and HT were administered was seen in the group treated with 2 Gy of radiation dose and 120 CEM43 of thermal dose. In this group RT preceding HT resulted in a cell viability of 13±2% on day 7 after treatment, whilst HT preceding RT resulted in a cell viability of 48±8%. Visual inspection of the spheroids showed an increased effect in the form of less growth, as well as flaking around the edges of the spheroids. Obtaining qualitative information on the effect that combined treatment had on spheroids using confocal microscopy was unsuccessful. Both using a live and dead cell staining kit, as well as PI staining proved ineffective in accurately visualizing the dead cells in spheroids.

Iron deficiency is considered the most significant nutritional deficiency worldwide, affecting billions of people. Iron deficiency leads to a higher prevalence of iron deficiency anaemia; a condition responsible for over 50,000 deaths annually. Since anaemia is considered a global health problem, various countermeasures have been implemented to battle this disease. One of these countermeasures is the supplementation of iron. Different iron compounds are employed for supplementation, including ferrous sulphate and ferrous fumarate. However, since only 15% of the supplemented iron is absorbed by the human body, the remaining 85% can cause various health-related issues. Due to heightened amounts of iron present in the duodenum, a disbalance in the human gut is induced, leading to e.g. excessive growth of Escherichia coli (E.coli). This study investigates the influence of 3 and 30 µM iron supplementation of ferric chloride and ferric sulphate; radiolabelled with Fe-55. These results have been reported in the form of growth curves, and the remaining amount of Fe-55 in the growth medium has been used to estimate iron uptakeby E.coli. No significant differences were observed between 3 and 30 µM iron supplementation of both compounds, indicating that iron concentration may not be considered as the main limiting growth factor of E.coli. Therefore, further research is recommended where the medium is analysed for the presence of other nutrients. Additionally, adjustments in the growth environment are recommended, to determine which growth conditions influence E.coli the most.

Cancer is one of the leading causes of death worldwide and the number of cases is expected to keep increasing in the next years. Even though nowadays most employed cancer treatments in clinical practice (surgery, chemo-, and radiotherapy) are effective, they are still associated with multiple limitations and side effects. The main pitfall stays in their non-specificity to tumour cells, which leads to affecting healthy tissues. Therefore, alternative treatments able to overcome the oncologic challenges of the current treatment regimens by specifically treating only the cancer cells, be minimally invasive, and limit short and long-term side effects are highly needed. As the number of patients diagnosed with cancer in incipient stages is constantly increasing, such alternative treatments are currently even more attractive. Due to the advances in nanotechnology, cancer nanomedicine is a fast-advancing field, employing nanoparticles to both diagnose and deliver therapy of cancer, namely, nanotheranostics. Nanobrachytherapy is the brachytherapy treatment delivered via injection of radioactive nanoparticles into the tumour. The great advantage is that nanobrachytherapy retains the characteristics of brachytherapy, such as precise and targeted dose delivery, while allowing a less invasive administration and a more uniform dose distribution in the tumour. However, the radio-resistance exhibited by the tumour cells can hinder the success of nanobrachytherapy, but the synergetic combination of cell damaging agents, as well as radioactivity and heating, is wellknown. Thermal treatments, such as hyperthermia, offer a hyperthermic radiosensitization making the tumour more susceptible to irradiation, while thermal ablation can serve as surgery replacement. Furthermore, thermal treatments can be delivered by injection of colloidal suspensions of magnetic nanoparticles (MNPs) in tumours and heating them via exposure to an externally applied alternating magnetic field. An additional advantage of such magnetic nanoparticles is their ability to ensure visualization via magnetic resonance imaging (MRI), a non-invasive technique, helpful in monitoring the treatment effects. This thesis aims to develop a nanotheranostic agent, able to deliver therapeutic effects via radiation and heating, with additional imaging via magnetic resonance imaging. We envision the nanotheranostic as a core-shell hybrid nanoparticle in the form of 103Pd:Pd/Fe-oxide. The palladium core is radiolabelled with 103Pd radioisotope, responsible for the required radiation dose, whereas the iron oxide coating ensures hyperthermia/thermal ablation and imaging…@en

Since lanthanides were discovered in the 18th century, they fascinate scientists due to their similarity in chemical behavior and versatility in physical properties. It is thus not surprising, that lanthanides are part of uncountable applications in modern industry, electronics, magnets, light sources and medical applications. Especially, as the lanthanide elements are not naturally occurring in living organisms, it is essential that the molecular dynamics and chemical as well as physical properties are understood in detail for in vivo applications. In this thesis, different lanthanide-containing compounds and nanoparticles were investigated to gain deeper knowledge of fundamental processes governing physical properties and synthetic procedures.@en

Female breast cancer is the second most common cancer worldwide. A promising novel technique to treat early-stage breast cancer is the use of thermo-brachy therapy. This thesis analysed the effect of the cancer biomarker glutathione (GSH) on the magnetic hyperthermal effects of iron oxide nanoparticles. The synthesised nanoparticles with a diameter of 10±4 nm have been coated with cysteine and treated with hydrogen peroxide leading to their aggregation upon cross-linking. As the GSH concentration is higher around cancerous cells (10 μM instead of 3 μM), these nanoparticles would be more monodispersed as GSH could break the disulfide bonds between the cysteines. Since aggregated nanoparticles exhibit lower heating efficiencies, it was assumed that this method would allow for (partially) selective heating of the tumour. The addition of 10 μM GSH resulted in an 11% higher heating efficiency compared to 3 μM GSH when analysing the coated nanoparticles in an alternating magnetic field. However, a GSH concentration of 10 mM (intracellular GSH concentration in cancerous cells) gave a decrease in heating efficiency. Another methodology was proposed as well. Uncoated nanoparticles were encapsulated in a ZIF-8 framework. This framework is less stable in environments with a lower pH value and higher GSH concentration, which is the case in the tumour microenvironment. This methodology gave a 27% higher heating efficiency in a tumour-related GSH concentration and pH value. The differences with both the cysteine and ZIF-8-based methods are yet too small to limit thermal damage to healthy tissue. Modelling the diffusion and its influence on the temperature change, gave very local heating near the injection site due to slow diffusion. Therefore, while this thesis provided insights into the heating efficiencies in tumour-related conditions with both methodologies, addressing the current limitations can lead to significant advancements.

Female breast cancer is the second most common cancer worldwide. A promising novel technique to treat early-stage breast cancer is the use of thermo-brachy therapy. This thesis analysed the effect of the cancer biomarker glutathione (GSH) on the magnetic hyperthermal effects of iron oxide nanoparticles. The synthesised nanoparticles with a diameter of 10±4 nm have been coated with cysteine and treated with hydrogen peroxide leading to their aggregation upon cross-linking. As the GSH concentration is higher around cancerous cells (10 μM instead of 3 μM), these nanoparticles would be more monodispersed as GSH could break the disulfide bonds between the cysteines. Since aggregated nanoparticles exhibit lower heating efficiencies, it was assumed that this method would allow for (partially) selective heating of the tumour. The addition of 10 μM GSH resulted in an 11% higher heating efficiency compared to 3 μM GSH when analysing the coated nanoparticles in an alternating magnetic field. However, a GSH concentration of 10 mM (intracellular GSH concentration in cancerous cells) gave a decrease in heating efficiency. Another methodology was proposed as well. Uncoated nanoparticles were encapsulated in a ZIF-8 framework. This framework is less stable in environments with a lower pH value and higher GSH concentration, which is the case in the tumour microenvironment. This methodology gave a 27% higher heating efficiency in a tumour-related GSH concentration and pH value. The differences with both the cysteine and ZIF-8-based methods are yet too small to limit thermal damage to healthy tissue. Modelling the diffusion and its influence on the temperature change, gave very local heating near the injection site due to slow diffusion. Therefore, while this thesis provided insights into the heating efficiencies in tumour-related conditions with both methodologies, addressing the current limitations can lead to significant advancements.

The goal of this research was to perform a 3D end-to-end test on a MR-linac to check the whole workflow using a clinical treatment plan. Dosimetric gel was used to obtain 3D spatial information, with the phantom in the same position for irradiation and scanning. In order to achieve this, fundamental elements of gel dosimetry needed to be investigated. In the MR-linac, irradiation is delivered in the presence of a permanent magnetic field. Therefore, the dosimetric response within a 1.5 T magnetic field should be validated. It is also important to investigate the time-dependence of the gel. It is preferable to read-out the gels within approximately one hour, so that the phantom does not have to be moved. Ideally, scanning and irradiation would be done at the same time, to see the dynamical dose delivery. The VIPAR gel was used for this research. The experiments demonstrated that R2 values for doses irradiated with magnetic field were the same as R2 values for the same dose irradiated without magnetic field. R2 values are still proportional to the dose. It was also shown that it is possible to scan the phantom within 20 minutes after irradiation. Sensitivity is at its highest after approximately 8 hours and stays stable afterwards, so scanning after 8 hours will improve the read-out accuracy. It was also possible to make a fit for the R2 versus time plots, which makes it possible to correct for change over time. The fit can be divided in two linear parts if time is plotted on a logarithmic scale, one fit for the time points before 7 hours, one for the time points after 7 hours. The partial doses acquired by the gel during radiation delivery were estimated. The equivalent R2 values then agreed with the extrapolated fit to within 4%. This is a good indication that dynamic gel (4D) dosimetry may be achievable. A protocol for a relative end-to-end test was also developed. From the preliminary results, it appeared that a relative end-to-end test can be performed with the read-out of gel within 1 hour. A new MR sequence needs to be developed. For this end-to-end test, the sequence needs to scan a larger volume with a higher resolution, therefore, the scan time will increase and real-time dosimetry will not be possible. Changing the MR sequence might also change the optimal irradiation-scanning interval and the R2 versus time curve. To perform absolute dosimetry, an extra calibration would be required.

Conventional early-stage breast cancer treatments such as surgery, chemotherapy and external radiotherapy despite their proven short-term efficacy tend to have adverse long-term physiological and psychological implications on the patients. This is primarily due to their inability to spare healthy tissue surrounding the tumor. Use of radioactive palladium (103Pd) seeds for producing localized effect with brachytherapy is already in practice. However, they are known to result in uneven dose distribution with creation of "hot spots" in the vicinity of seed implants. Alternatively, conventional thermal ablation or hyperthermia treatments using mechanical or electromagnetic systems also have difficulties with localizing the thermal effect to the target region. Composite and biocompatible palladium- (Ultra-small) superparamagnetic iron oxide nanoparticles (PdO-USPION) on the other hand have the potential to diffuse throughout the tumor ensuring relatively more uniform dose distribution and improve the ease of clearance from the biological system without causing long-term side effects. Magnetic property of (Ultra small) superparamagnetic iron oxide nanoparticles can be exploited to induce and deliver heat energy to kill cancerous cells upon exposure to Alternating magnetic field (AMF) and generate contrast enhanced magnetic resonance images (MRI) by subjecting them to static magnetic fields. Heating and contrast enhancement ability depends on the physical, chemical and magnetic properties of nanoparticles which in turn rely on the synthesis method. In this research, spark ablation technology is employed to produce the nanoparticles from metallic electrodes. Although, for the purpose of the current research, inactive palladium is used to reduce complexity given that the synthesis of PdO-USPIONs with this synthesis method is employed for the first time through this research. Inter-metallic Pd-Fe are generated in the spark discharge system and captured in an aqueous media resulting in (oxidized intermetallic palladium ultra-small superparamagnetic iron oxide nanoaparticles) PdO-USPIONs. Since the synthesis method is relatively new, major component of the research deals with investigating the influence of system parameters on production of nanoparticles. Characterization results associated with USPIONs and PdO-USPIONs are thoroughly analyzed to optimize the setup to generate tunable PdO-USPIONs and to evaluate their performance as thermal and contrast agents. Use of citric acid as surfactant to lower agglomeration resulting in improved T1 relaxation behavior is also studied.

Cancer is still one of the most serious diseases for humanity. Internal radiation therapy with alpha particles is a promising method to battle cancer, but it is currently limited to easily accessible tumors. A more advanced and selective method for alpha radiation therapy that will have better outcomes, fewer side effects, and higher efficacy is needed. A promising alternative is a two-step approach in which super paramagnetic iron oxide nanoparticles (SPION’s) are first delivered to the cancer cells and then receive the radiotherapeutics in a targeted manner. One of the most important conditions for this strategy is retention of SPIONs at the cell membranes in order to ensure in vivo click-reaction between the azide-functionalized nanoparticles and cyclooctyne-conjugated radiotracer. The goal of this study is to observe the behavior of two types of SPION’s on the glioblastoma multiforme (GBM) cancer cell line U87 and to verify whether the nanoparticles are internalized by the cancer cells or stay on the outside of the membrane. This has been done by growing U87 spheroids, incubating them with the nanoparticles, performing a cryosection on these spheroids, photographing the slices of the cryosection with a fluorescence microscope and analyzing the iron-content of the slices with inductively coupled plasma optical emission spectrometry (ICP-OES). The second part of the study concerns the preliminary in vitro click-reaction of the nanoparticles by adding beta emitter 177Lu coupled to DOTA-cyclooctyne complex to spheroids that were incubated with the nanoparticles. The fluorescence microscope analysis concludes that the nanoparticles functionalized with polyethylene glycol (PEG) penetrate the U87 spheroids less than the nanoparticles without PEG. The azide click reaction between the nanoparticles and 177Lu-DOTA-cyclooctyne complex was observed and showed more cell damage to the spheroid incubated with nanoparticles then the spheroids that were only treated with 177LuDOTA-cyclooctyne.

The recently discovered negative health effects of Gd(III) complexes based magnetic resonance imaging (MRI) contrast agents have stirred interest into the development of alternatives based on Mn(II) complexes due to its biocompatibility, and hence less health concerns. One important aspect of investigation on contrast agents in general, and Mn(II) complexes in particular, is an accurate determination of their concentrations in solution. Therefore, in this study a known approach of determining the concentration of paramagnetic species in solution, via the Bulk Magnetic Susceptibility (BMS) shift, is applied for Mn(II) and the feasibility of the method is evaluated. A proof of principle using Gd-DOTP concentrations in the range 0.7 – 6.6 mM Gd(III) to verify the applied BMS method was successfully performed. Subsequently, BMS measurements were performed with MnDOTA as well as free Mn(II) and the obtained concentrations were verified with ICP-OES. A good agreement between the two methods was found within the concentration range of 0.4 - 15.1 mM Mn(II) for both MnDOTA and free Mn(II). Although, BMS measurements showed slightly higher concentrations than those found by ICP-OES for both Mn-DOTA and free Mn(II). Additionally, the deviations between ICP-OES and BMS were greater for free Mn(II) compared to Mn-DOTA. Experiments for determining the accuracy of the BMS method showed a 2.5% upward systematic error. The random error decreased from 20% for 0.27 mM Mn(II) to almost 0% for 4.0 mM Mn(II) and higher concentrations. In conclusion, the BMS method proved to be accurate for determining Mn(II) concentrations and has promising prospects for future research into Mn(II) complexes. However, deviations between ICP-OES and BMS measurements need further investigations. Key words: Bulk Magnetic Susceptibility shift, contrast agent, Mn(II) complexes.

Neutron capture therapy (NCT) is a binary form of radiotherapy that utilizes the high cross section of some nonradioactive elements, in particular boron-10 and gadolinium-157, for thermal neutron capture. The energetic charged particles (electrons and alpha particles) released in the capture reaction cause a high localized deposition. For almost a century, NCT has been regarded as the holy grail of targeted radiotherapy, but its superiority over other available treatments has not been demonstrated so far. Limitations included amongst others a poor and non-selective biodistribution of Boron carrier molecules, the limited availability of suitable neutron sources, a lack of tools for patient selection and individualized dosimetry, and a shortage of properly conducted clinical trials.

Cancer is one of the leading causes of death in the world. Because of this, there are many novel methods to treat it being currently researched. One of the answers from the nuclear medicine perspective is Radionuclide Therapy, where alpha or beta emitters are applied, with the goal to selectively irradiate tumours in the body. A promising radionuclide researched for Radionuclide Therapy is Holmium-166, which is a beta emitter with a short half-life of 26.8 hours, which is useful for the treatment of large metastases. A method to ensure an effective treatment with Holmium-166 is the use of a Dysprosium-166/Holmium-166 in vivo generator, as the dose delivered to patients per administered dose is two times higher with the in vivo generator rather than the direct use of Holmium-166. However, carriers used in Radionuclide Therapy, which usually involve the formation of a chelator-metal complex are not effective for the in vivo generator due to the release of auger electrons during the decay process leading to the destruction of the chelator-metal complex. A promising non-chelator method designed by Liu et al. at the Applied Radiation and Isotopes research team at TU Delft involves the radiolabelling of micelles. This thesis sought to evaluate the use of micelles, radiolabelled with this mechanism as an effective carrier for the Dysprosium-166/Holmium-166 in vivo generator. For this, micelles made of Polycaprolactone-block-Polyethylene Oxide and Polylactic Acidblock-Polyethylene oxide were evaluated based on the obtained radiolabelling efficiency and their stability when challenged with diethylenetriaminepentaacetic acid (DTPA). The results in this study show that the radiolabelling method used to encapsulate Dysprosium relies on the diffusion of Dysprosium hydroxides into the micelle core, followed by precipitation as the right concentration is reached inside the micelles. The best results obtained by this study occur when radiolabelling Polylactic Acid-block-Polyethylene Oxide (PLA-PEO) micelles, and then adding phosphate ions at a concentration of 3 × 10−8𝑀, 30 minutes after the addition of the active Dysprosium. Although more tuning is required on the radiolabelling mechanism to make micelles the most effective carriers of the 166Dy/166Ho in vivo generator, promising first steps were made to begin this process